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WO2005071404A1 - Procede de diagnostic de cancer colorectal et reactifs de mise en oeuvre - Google Patents

Procede de diagnostic de cancer colorectal et reactifs de mise en oeuvre Download PDF

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Publication number
WO2005071404A1
WO2005071404A1 PCT/AU2005/000077 AU2005000077W WO2005071404A1 WO 2005071404 A1 WO2005071404 A1 WO 2005071404A1 AU 2005000077 W AU2005000077 W AU 2005000077W WO 2005071404 A1 WO2005071404 A1 WO 2005071404A1
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Prior art keywords
seq
methylation
colorectal cancer
nucleic acid
sequence
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PCT/AU2005/000077
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English (en)
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Maija Kohonen-Corish
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Garvan Institute Of Medical Research
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Priority claimed from AU2004900340A external-priority patent/AU2004900340A0/en
Application filed by Garvan Institute Of Medical Research filed Critical Garvan Institute Of Medical Research
Priority to EP05700109A priority Critical patent/EP1711813A4/fr
Priority to CA002553540A priority patent/CA2553540A1/fr
Priority to US10/586,978 priority patent/US20070243531A1/en
Priority to AU2005206590A priority patent/AU2005206590A1/en
Publication of WO2005071404A1 publication Critical patent/WO2005071404A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/154—Methylation markers

Definitions

  • the present invention relates to methods for the diagnosis and treatment of cancer, particularly colorectal cancer. More particularly, the present invention provides methods for the diagnosis and treatment of colorectal cancer in a subject comprising determining the degree of methylation of nucleic acid that regulates expression of a tumor suppressor gene in a sample from the subject.
  • derived from shall be taken to indicate that a specified integer is obtained from a particular source albeit not necessarily directly from that source.
  • nucleotide and amino acid sequence information prepared using Patentln Version 3.1, presented herein after the claims.
  • Each nucleotide sequence is identified in the sequence listing by the numeric indicator ⁇ 210> followed by the sequence identifier (e.g. ⁇ 210>1, ⁇ 210>2, ⁇ 210>3, etc).
  • the length and type of sequence (DNA, protein (PRT), etc), and source organism for each nucleotide sequence are indicated by information provided in the numeric indicator fields ⁇ 211>, ⁇ 212> and ⁇ 213>, respectively.
  • Nucleotide sequences referred to in the specification are defined by the term "SEQ ID NO:", followed by the sequence identifier (eg. SEQ ID NO: 1 refers to the sequence in the sequence listing designated as ⁇ 400>1).
  • nucleotide residues referred to herein are those recommended by the IUPAC-IUB Biochemical Nomenclature Commission, wherein A represents Adenine, C represents Cytosine, G represents Guanine, T represents thymine, Y represents a pyrimidine residue, R represents a purine residue, M represents Adenine or Cytosine, K represents Guanine or Thymine, S represents Guanine or Cytosine, W represents Adenine or Thymine, H represents a nucleotide other than Guanine, B represents a nucleotide other than Adenine, V represents a nucleotide other than Thymine, D represents a nucleotide other than Cytosine and N represents any nucleotide residue.
  • Cancer is a multi-factorial disease and major cause of morbidity in humans and other animals, and deaths resulting from cancer in humans are increasing and expected to surpass deaths from heart disease in future.
  • Carcinomas of the lung, prostate, breast, colon, pancreas, and ovary are major contributing factors to total cancer death in humans. With few exceptions, metastatic disease from carcinoma is fatal. Even if patients survive their primary cancers, recurrence or metastases are common.
  • Colorectal cancer is the second most frequently diagnosed malignancy in the United States as well as the second most common cause of cancer death.
  • the five-year survival rate for patients with colorectal cancer detected in an early localized stage is about 92%. However, only about 37% of colorectal cancer is diagnosed at an early localized stage.
  • the survival rate of patients suffering from colorectal cancer drops to about 64% if the cancer is allowed to spread to adjacent organs or lymph nodes, and to 7% in patients with distant metastases.
  • Diagnosis of colorectal cancer is known to utilize screening assays for fecal occult blood, sigmoidoscopy, colonoscopy and double contrast barium enemas.
  • the prognosis of colorectal cancer is directly related to the degree of penetration of the tumor through the bowel wall and the presence or absence of nodal involvement. As a consequence, early detection and treatment of colorectal cancer are highly desirable.
  • colorectal cancers may develop by distinct pathways having different clinical, pathological and molecular characteristics. For example, chromosomal instability or microsatellite instability (MSI) may occur.
  • MSI microsatellite instability
  • CpG islands in D ⁇ A occurs to varying degrees in colorectal cancers having a high frequency of microsatellite instability (Toyota et al, Proc. Natl Acad. Sci USA 97, 710- 715, 2000), and in serrated adenoma (Park et al, Am. J. Pathol 162, 815-822, 2003), and at low frequency in certain hyperplastic polyps (Chan et al, Am. J. Pathol. 160, 529-536, 2002).
  • CpG island methylation phenotype may indicate microsatellite instability.
  • MINTs the degree of methylation of a plurality of anonymous CpG island sequences (MINTs), not a single marker, is determined.
  • MINTs i.e., MINT 1, MINT 2, MINT 12 and MINT 31
  • MINT 1 MINT 1, MINT 2, MINT 12 and MINT 31
  • MINT 1 MINT 1, MINT 2, MINT 12 and MINT 31
  • MINT 1 MINT 1, MINT 2, MINT 12 and MINT 31
  • MINTs are sufficiently sensitive and reliable to determine a CIMP when used together (Toyota et al, Proc. Natl Acad. Sci USA 97, 710-715, 2000; Hawkins et al, Gastroenterol 122, 1376-1387, 2002; Whitehall et al, Cancer Res. 62, 6011-6014, 2002).
  • these MINTs are not used routinely to diagnose/prognose colorectal cancer, let alone at an early stage.
  • Treatment regimens are determined by the type and stage of the cancer, and include surgery, radiation therapy and/or chemotherapy. Recurrence following surgery (the most common form of therapy) is a major problem and is often the ultimate cause of death.
  • a stage 0 colorectal cancer or "carcinoma in situ" is a pre-cancerous condition, usually found in a polyp.
  • Epithelial lesions with glandular serration include aberrant crypt foci (ACF), hyperplastic polyps (HP), mixed polyps (MP), serrated adenomas (SA) (e.g., Longacre et al, Am. J. Surg. Pathol. 14, 524-537, 1990) and sessile serrated adenomas (SSA; Arthur et al, J. Clin. Pathol. 21, 735-743, 1968). It is generally thought that MP and SA arise from HP.
  • ACF aberrant crypt foci
  • HP hyperplastic polyps
  • MP mixed polyps
  • SA serrated adenomas
  • SSA sessile serrated adenomas
  • hyperplastic polyposis may represent the most severe manifestation of a predisposition to produce large serrated polyps with an increased risk of malignancy.
  • Wynter et al, Gut 53, 573-580, 2004 suggest that ACF and traditional HP, are non-progressing lesions having little or no potential for neoplastic transformation, however those variant HP in which methylation of at least 3-4 CIMPs occurs may progress to SA or MP and eventually colorectal cancer.
  • Wynter et al. also proposed that whether or not such CIMP-high polyps progressed depends on the additional presence of a BRAF gene mutation (or possibly a K-ras mutation), and loss of function of the DNA repair genes MGMTandMLHl.
  • HP representing hyperplastic polyposis are morphologically distinguishable by size, architecture, differentiation, and proliferation, from traditional HPs localized to the distal colon and rectum. (Torlakovic et al, Gastrolenterol. 110, 748-755, 1996).
  • numbers of polyps and anatomical location within the colorectum are not indicators of a predisposition for malignancy, because those HPs giving rise to malignancy are not localized.
  • serrated polyps e.g., certain HPs, SA and MP
  • early stage colorectal cancer e.g., hyperplastic polyposis.
  • a K-ras mutation has been detected at high frequency in ACF, (Chan et al, Am. J. Pathol. 160, 1823-1830, 2002; Jen et al, Cancer Res 54, 5523-5526, 1994), however occurs at a reduced frequency in hyperplastic polyposis (Otori et al, Gut 40, 660-663, 1997), SA (Ajioka et al, Gut 42, 680-684, 1998; Sawyer et al, Gut 51, 200-206, 2002), and sporadic colorectal cancer with high level MSI (Samowitz et al, Am. J. Pathol. 755, 1517-1524, 2001). Mutation or loss of the tumor suppressor genes p53 and Smad4/DPC4A may also accompany the transition from normal colonic mucosa to metastatic carcinoma.
  • Stage 1(A) colorectal cancer A stage 1(A) colorectal cancer is one that is confined to the lining of the colon and does not penetrate the wall of the colon into the abdominal cavity, and has not spread to any adjacent organs or local lymph nodes and cannot be detected in other locations in the body, as determined following surgical removal of the tumor.
  • Stage 1(A) colorectal cancer is generally curable by surgical resection. Depending on features of the cancer under the microscope, approximately 90% of patients are cured without evidence of cancer recurrence following treatment with surgery alone. A minority of patients with stage I colorectal cancer may experience recurrence, partly as a consequence of undetectable micro metastases outside the colon.
  • a stage II (B) colorectal cancer is one that has penetrated the wall of the colon into the abdominal cavity, but does not invade any of the local lymph nodes and cannot be detected in other locations in the body, as determined by histopathology of resected colon.
  • the tumor has penetrated the wall of the colon into the abdominal cavity and invaded any of the local lymph nodes, but cannot be detected in other locations in the body, as determined by histopathology of resected colon.
  • a stage IV (D) colorectal cancer is one that has metastasized to distant locations in the body, which may include the liver, lungs, bones or other sites.
  • stage IV colon cancer Patients diagnosed with stage IV colon cancer have been perceived to have few treatment options. Certain patients, however, can still be cured of their cancer, and others derive significant benefit from additional treatment. Patients with stage IV colon cancer can be broadly divided into two groups: those with cancer spread that is localized to a single site and those with more widespread cancer.
  • HNPCC Hereditary Non- Polyposis Colorectal Cancer
  • HNPCC accounts for about 2% of colorectal cancer cases, and is characterized by the tendency to early onset of cancer and the development of other cancers, particularly those involving the endometrium, urinary tract, stomach, and biliary system. HNPCC is thought to be a consequence of mutations of one or more genes in the DNA mismatch repair (MMR) pathway, in particular the MSH2 and MLHl genes. Loss of MMR activity is believed to contribute to cancer progression by virtue of cells accumulating other gene mutations and deletions, such as loss of the BAX gene which controls apoptosis, and the TGF ⁇ 2 receptor gene which controls cell growth. Familial Adenomatous Polyposis (FAP)
  • MMR DNA mismatch repair
  • FAP is characterized by the early development of multiple colorectal adenomas that progress to cancer at a mean subject age of 44 years.
  • FAP is believed to be caused by an inherited mutation in the adenomatous polyposis coli (APC) gene, thereby resulting in the loss of orderly replication, adhesion, and migration of colonic epithelial cells and leading to polyposis.
  • APC adenomatous polyposis coli
  • Much attention has focussed on the detection of inherited and somatic mutations of the APC gene in colorectal cancer, especially as a diagnostic for FAP.
  • U.S. Pat. No 5,648,212 incorporated by reference disclosed several mutations within the APC gene as being diagnostic of colorectal cancer, and teach methods of diagnosis of colorectal cancer based upon those mutations.
  • Ulcerative colitis predisposes affected individuals to about a 20-fold increased risk of developing cancer, associated with mutations in the p53 gene which may occur early, appearing even in histologically-normal tissue.
  • the progression of the disease from ulcerative colitis to dysplasia/carcinoma without an intermediate polyp state suggests a high degree of mutagenic activity resulting from the exposure of proliferating cells in the colonic mucosa to the colonic contents.
  • the inventors sought to develop new markers for the sensitive, reliable and straightforward diagnosis of colorectal cancer, especially for determining those early stage polyps of clinical significance or having transformation or neoplastic potential.
  • MCC colorectal cancer
  • aberrant hyper methylation of the MCC gene occurs in at least about 50% of cases. This aberrant methylation is also correlated with reduced expression of the gene in subjects suffering from colorectal cancer.
  • the present inventors showed that the promoter region of the MCC gene, and still more particularly the region between about position -600 and about position -139 relative to the translation start site of the MCC gene, is hyper methylated in samples derived from colorectal cancer subjects, and more preferably from about position -394 to about position -139 relative to the translation start site of the MCC gene.
  • 26 CpG sites were shown to be the most reliable indicators of reduced MCC gene expression or silencing.
  • Methylation of MCC also correlates positively with a number of climcopathological variables in stage C colorectal cancer, in particular tumor grade i.e., degree of differentiation e.g., poorly differentiated grade 3 or moderately differentiated grade 2 or well-differentiated grade 1, and number of malignant lymph nodes, there being higher grade tumors and a higher number of malignant lymph nodes at stage C in subjects having hypermethylation in the MCC gene. Additionally, stage C tumors in the proximal colon are more frequently associated with MCC methylation suggesting to the inventors that they may arise from variant HPs or SSA rather than traditional HPs.
  • tumor grade i.e., degree of differentiation e.g., poorly differentiated grade 3 or moderately differentiated grade 2 or well-differentiated grade 1
  • stage C tumors in the proximal colon are more frequently associated with MCC methylation suggesting to the inventors that they may arise from variant HPs or SSA rather than traditional HPs.
  • the inventors designed novel nucleic acid probes for specifically detecting hyper methylated MCC gene promoter fragments in cell lines derived from colorectal cancer subjects, and showed that these nucleic acid probes also successfully detected hyper methylated MCC gene promoter fragments in samples derived from at least about 40% of colorectal patients having stage C colorectal cancers, but not in samples derived from comparable samples from normal tissues of the same subjects.
  • MCC is hypermethylated in a significant proportion of HP, e.g., at least about 80% of HP and more preferably at least about 90% of HP or about 100% of HP.
  • methylation of MCC is not necessarily correlated to microsatellite instability. This contrasts with previous studies showing methylatioin of the DNA mismatch repair gene hMLHl.
  • MCC methylation as a marker for a number of diagnostic applications, including: (i) detecting a precancerous lesion; (ii) determining a predisposition for neoplastic transformation in a subject having colorectal polyps; and/or (iii) determining the likelihood of progression of a colorectal polyp to SA and/or MP and/or colorectal cancer of any stage (e.g., progression of HP to SA and/or MP); and/or (iv) determining the malignant potential of a colorectal polyp; and/or (v) diagnosing hyperplastic polyposis; and/or
  • the present invention provides a method of diagnosing colorectal cancer or hyperplastic polyposis in a subject, said method comprising determining the degree of methylation of nucleic acid that regulates expression of an MCC gene in a sample derived from the subject, wherein an enhanced degree of methylation in the sample relative to a suitable control sample is indicative of colorectal cancer or hyperplastic polyposis.
  • the present invention also provides a method for determining a predisposition for neoplastic transformation in a subject having colorectal lesions or polyps said method comprising determining the degree of methylation of nucleic acid that regulates expression of an MCC gene in a sample derived from a lesion or polyp wherein an enhanced degree of methylation in the sample relative to a suitable control sample is indicative of a predisposition for neoplastic transformation in the subject.
  • predisposition for neoplastic transformation in a subject having colorectal polyps shall be taken to mean that a colorectal polyp has neopolastic or malignant potential and/or is likely to progress to SA and/or MP and/or colorectal cancer of any stage. Accordingly, the term "method of determining a predisposition for neoplastic transformation in a subject having colorectal polyps" applies mutatis mutandis to such applications and indications.
  • the diagnostic/predictive tests described hererin are useful for monitoring the efficacy of surgical resection, chemotherapy, radiation therapy undetaken for the purposes of treating a colorectal cancer, because the treatment should ablate the aberrant methylation pattern of a colorectal sample taken after treatment. It will also be apparent that the invention is useful for determining recurrence of polyps having neoplastic potential or colorectal cancer.
  • the present invention provides a method of monitoring the efficacy of treatment for colorectal cancer or hyperplastic polyposis in a subject, said method comprising treating a subject in need of treatment for colorectal cancer or hyperplastic polyposis and determining the degree of methylation of nucleic acid that regulates expression of an MCC gene in a sample derived from the subject, wherein a degree of methylation in the sample comparable to that for a suitable control sample is indicative of effective treatment.
  • the present invention provides a method of monitoring the efficacy of treatment for colorectal cancer or hyperplastic polyposis in a subject, said method comprising treating a subject in need of treatment for colorectal cancer or hyperplastic polyposis and determining the degree of methylation of nucleic acid that regulates expression of an MCC gene in a sample derived from the subject, wherein a hyper methylation of the MCC promoter in the sample compared to the level of methylation for a suitable control sample indicates that treatment is not effective.
  • Preferred embodiments of the present invention are directed to multianalyte tests wherein the level of methylation of MCC and another gene selected from the group consisting of HLTF, APC, pi 6 m ⁇ 4a , pi ' ⁇ SF , HPP1, hMLHl, MGMT, and combinations thereof is also determined. Accordingly, hypermethylation of MCC and the other analyte is indicative of colorectal cancer or hyperplastic polyposis or a predisposition of a lesion or polyp to neoplastic transformation.
  • the level of methylation of MCC and a CpG island methylation phenotype is determined using CIMP markers known in the art e.g., MINT 1, MINT 2, MINT 12 and MINT 31.
  • CIMP markers known in the art e.g., MINT 1, MINT 2, MINT 12 and MINT 31.
  • Hypermethylation of MCC in combination with methylation of 3-4 CIMP markers is indicative of colorectal cancer or hype ⁇ lastic polyposis or a predisposition of a lesion or polyp to neoplastic transformation.
  • the level of methylation of MCC and a mutation in the APC gene or DCC gene are determined.
  • Hypermethylation of MCC in combination with a deletion or point mutation of APC or DCC are indicative of of colorectal cancer or hype ⁇ lastic polyposis or a predisposition of a lesion or polyp to neoplastic transformation.
  • Such multianalyte formats are particularly suited for all diagnostic and predictive applications described herein.
  • the present invention also provides a method of diagnosing colorectal cancer or hype ⁇ lastic polyposis in a subject or determining a predisposition for neoplastic transformation in a subject having colorectal lesions or polyps, said method comprising (i) determining the degree of methylation of nucleic acid that regulates expression of an MCC gene in a sample derived from the subject; and (ii) determining the degree of methylation of at least one nucleic acid selected from the group consisting of a nucleic acid regulating expression o ⁇ HLTF, a nucleic acid regulating expression of APC, a nucleic acid regulating expression of pl6 m ⁇ 4a , a nucleic acid regulating expression of pl4 RF , a nucleic acid regulating expression of HPP1, a nucleic acid regulating expression of hMLHl, a nucleic acid regulating expression of MGMT, and combinations thereof in a sample derived from the subject, wherein an enhanced degree of
  • the present invention also provides a method of diagnosing colorectal cancer or hype ⁇ lastic polyposis in a subject or determining a predisposition for neoplastic transformation in a subject having colorectal lesions or polyps, said method comprising (i) determining the degree of methylation of nucleic acid that regulates expression of an MCC gene in a sample derived from the subject; and (ii) determining a mutation such as a point mutation or deletion in a gene selected from the group consisting of APC and DCC, wherein an enhanced degree of methylation of the nucleic acid at (i) and a mutation at (ii) relative to a suitable control sample is indicative of colorectal cancer or hype ⁇ lastic polyposis or a predisposition for neoplastic transformation of a lesion or polyp in the subject.
  • the present invention also provides a method of diagnosing colorectal cancer or hype ⁇ lastic polyposis in a subject or determining a predisposition for neoplastic transformation in a subject having colorectal lesions or polyps, said method comprising (i) determining the degree of methylation of nucleic acid that regulates expression of an MCC gene in a sample derived from the subject; and (ii) determining the CpG island methylation phenotype of a sample derived from the subject using at least three MINT markers, wherein an enhanced degree of methylation of the nucleic acid at (i) and methylation of at least three MINT markers at (ii) relative to a suitable control sample is indicative of colorectal cancer or hype ⁇ lastic polyposis or a predisposition for neoplastic transformation of a lesion or polyp in the subject.
  • the present invention provides reagents for performing the diagnostic/predictive tests, in particular an isolated nucleic acid probe or primer that is capable of selectively hybridising to a region of the MCC gene promoter that is hyper methylated in a colorectal cancer, wherein said region comprises or is contained within nucleotide residues from about position 284 to about position 403 or about position 404 of SEQ ID NO: 3 or SEQ ID NO: 17 or SEQ ID NO: 24.
  • Figure 1 is a copy of a schematic representation showing the double-stranded nucleotide sequence of the MCC gene promoter and the positions of CpG islands therein.
  • CpG islands in the promoter are indicated by the shaded boxes that occur within the bracketed region to the left of the figure.
  • the translation initiation site in the MCC gene is also indicated by the shaded ATG and the arrow.
  • Figure 2 is a copy of a schematic representation showing the double-stranded nucleotide sequence of the MCC gene promoter from a variety of colon cancer cells following treatment with bisulphite.
  • CpG islands in the promoter that are protected from mutagenesis by bisulphite are indicated by the shaded boxes.
  • Filled circles indicate cytosine residues within CpG islands for which methylation was observed in a colorectal cancer cell line, wherein the number of circles indicate the number of colorectal cancer cell lines which exhibit methylation at the indicated position.
  • the positions of the region between positions -542 to -392 relative to the start of translation, showing variable levels of CpG methylation in different cell lines, are indicated at the left of the figure.
  • the position of a region of the MCC gene promoter, from position - 304 to position -139, that is hyper methylated in 6/13 colon cancer cell lines tested, is also indicated at the left of the figure.
  • the boxed nucleotide sequences indicate positions for annealing of sequencing primers (light boxes) and the position of a section of DNA from position -354 to position -320 that is resistant to bisulphite treatment albeit not necessarily as a consequence of methylation (heavy boxed sequence).
  • Figure 3 is a copy of a photographic representation showing reduced expression of MCC as determined by RT-PCR in colon cancer cells HCT15 and LS411N compared to the ovarian cancer cell line TOV21G.
  • RT-PCR was performed to amplify MCC- encoding mRNA from the cell lines indicated at the top of each lane.
  • the panel to the right of the figure is a control showing ALAS-1 expression in all three cell lines.
  • Data show that expression of MCC mRNA is reduced in the colon cancer cell lines HCT15 and LS41 IN, compared to the ovarian cancer cell line TOV21G, however expression of ALAS-1 is similar in all three cell lines.
  • Figure 4 is a copy of a schematic representation showing the double-stranded nucleotide sequence of the MCC gene promoter and the positions of primers for methylation-specific PCR to detect methylated CpG islands therein.
  • the double- stranded nucleotide sequence of the MCC gene promoter from a variety of colon cancer cells following treatment with bisulphite is indicated, showing the positions of CpG islands in the promoter that are protected from mutagenesis by bisulphite (shaded boxes) and the degree of methylation (filled circles) as for the legend to Figure 2.
  • the sequences of forward and reverse primers dasheavy shaded sequences for amplification of a methylated region of the MCC gene promoter that is diagnostic for colon cancer are also indicated.
  • the arrows indicate the direction of extension from the primers furing amplification such that the intervening sequence between the primers is amplified.
  • These primers only amplify MCC promoter sequences from cell lines in which the CpG islands within the priming sites are methylated and, as a consequence, protected from mutagenesis by bisulphite. Amplified fragments can be sequenced to confirm methylation.
  • Figure 5 is a copy of a photographic representation showing the products of a methylation-specific PCR assay in several different cell lines as indicated at the top of each lane. Methylation-specific PCR was performed as described using the primers indicated in Figure 4.
  • the cell lines designated Lispl, Co-115, LS513, KM12SM, LS411N, HCT15 and SW480 are colorectal cancer cell lines. Data indicated that methylation-specific PCR correlates with bisulphite sequencing results.
  • Figure 6 is a copy of a photographic representation showing methylation of the MCC gene promoter in primary colorectal tumors (C), normal tissue (N) and cancer of the lymph node (CLN).
  • Numbers at the top of colorectal tumor lanes refer to patients from whom the tumors were derived.
  • the upper panels indicate results for methylation- specific PCR of the MCC gene promoter. Methylation of the MCC gene promoter was observed in primary cancer tissues from patient samples numbered 1, 2, 3, 4, 6, and 7 and also in the lymph node cancer from patient number 6. The lower panel indicates results wherein the MYOD gene, which is known to be independent of methylation, was amplified from the samples indicated at the top of the figure.
  • Figure 7 is a graphical representation showing specific amplification of methylated MCC promoter using real-time PCR in a quantitative TaqMan assay format.
  • Figure 8 is a graphical representation showing amplification of MYOD gene sequences following treatment with bisulphite using real-time PCR in a quantitative TaqMan assay format, used as a control for the integrity of DNA samples in assays performed to determine methylation of MCC promoter sequences.
  • Figure 9 is a schematic representation showing methylation of MCC, pi 6 and APC gene promoters in seven representative hype ⁇ lastic polyps (HPl, HP2, HP3, HP4, HP5, HP6 and HP7) and twelve representative adenomas (AD1, AD2, AD3, AD4, AD5, AD6, AD7, AD8, AD9, AD10, AD11 and AD12).
  • the shading indicates methylation as determined using methylation specific PCR.
  • One aspect of the present invention provides a method of diagnosing colorectal cancer in a subject, said method comprising determining the degree of methylation of nucleic acid that regulates expression of an MCC gene in a sample derived from the subject, wherein an enhanced degree of methylation in the sample relative to a suitable control sample is indicative of colorectal cancer.
  • the term “diagnosis”, and variants thereof, such as, but not limited to “diagnose”, “diagnosed” or “diagnosing” shall not be limited to a primary diagnosis of a clinical state, however should be taken to include any primary diagnosis of a clinical state.
  • the "diagnostic assay” formats described herein are equally relevant to assessing the remission of a patient, or monitoring disease recurrence, or tumor recurrence, such as following surgery, radiation therapy, adjuvant therapy or chemotherapy, or determining the appearance of metastases of a primary tumor. All such uses of the assays described herein are encompassed by the present invention.
  • colonal cancer shall be taken to include a chromosomal abnormality in a cell of the colon and/or rectum of a human or non- human mammal characterized by deletion, reduced expression or silencing of the region linked to map position 5q21-22 of the human genome or corresponding position in the genome of a non-human mammal, or a condition selected from the group consisting of Stage 0 (carcinoma in situ) especially characterized by the presence of epithelian lesions such as serrated lesions (e.g., ACF, traditional HP, variant HP, MP, SA or SSA), stage A colorectal cancer, stage B colorectal cancer, stage C colorectal cancer, stage D colorectal cancer, or hype ⁇ lastic polyposis or a tumor arising therefrom including carcinoma, adenocarcinoma, sarcoma, lymphoma, carcinoid or gastrointestinal stromal tumor.
  • Stage 0 carcinoma in situ
  • epithelian lesions e.g., A
  • colon cancer as used herein shall be taken to include an early or developed tumor of the colon and/or rectum, such as, for example, any metastases thereof.
  • MCC gene shall be taken to mean a nucleic acid, including any genomic gene, that is linked to or positioned at map position 5q21-q22 of the human genome, or any mRNA transcript thereof, or any genomic gene or mRNA transcript from a human or non-human animal that comprises a nucleotide sequence having at least about 80% identity to the sequence of a human MCC gene transcript as set forth in SEQ ID NO: 1.
  • MCC gene As will be known to those skilled in the art, mutations within the 5q21-22 region of the human genome are common in colorectal cancer and, as a consequence, the term “MCC gene” clearly encompasses any mutation of a wild- type or naturally-occurring MCC gene, including any nucleic acid deletion thereto or any nucleic acid substitution or insertion therein.
  • MCC gene shall also be taken to include a gene encoding a peptide, polypeptide or protein from a human or non-human animal having an amino acid sequence that is at least about 80% identical to the sequence set forth in SEQ ID NO: 2.
  • nucleotide sequence set forth in SEQ ID NO: 1 relates to the mRNA transcript of the human MCC gene (Kinzler et al, Science 251, 1366-1370, 1991).
  • the percentage identity to SEQ ID NO: 1 or 2 is at least about 85%, more preferably at least about 90%, even more preferably at least about 95% and still more preferably at least about 99%.
  • the MCC gene is a human MCC gene.
  • nucleotide identities and similarities are calculated using the BLAST or BESTFIT programs of the Computer Genetics Group, Inc., University Research Park, Madison, Wisconsin, United States of America, to maximize the number of identical/similar amino acids and to minimize the number and/or length of sequence gaps in the alignment.
  • MCC gene shall also be taken to include any variant of SEQ ID NO: 1, such as, for example, a variant that encodes a variant of the amino acid sequence set forth in SEQ ID NO: 2 wherein arginine at position 506 is substituted for glutamine (i.e., MCC R506Q) and/or wherein lysine at position 233 is substituted for hreonine (i.e., MCC K233T) and/or wherein glutamate at position 234 is substituted for alanine (i.e., MCC E234A).
  • nucleic acid that regulates expression of an MCC gene shall be taken to mean a nucleic acid operably linked to the coding region of said gene in a human or animal subject wherein said nucleic acid confers, promotes or otherwise modulates expression of said gene in a cell of the subject, including any enhancer element, silencer element, or promoter, preferably contained within the 5 -untranslated region of the MCC gene.
  • the term "acid that regulates expression of an MCC gene” does not extend to a heterologous promoter that is used to regulate expression of an MCC gene in a cell that has been transformed or transfected with a recombinant vector comprising the heterologous promoter operably linked to the MCC gene.
  • promoter includes the transcriptional regulatory sequences of a classical genomic gene, including the TATA box which is required for accurate transcription initiation, with or without a CCAAT box sequence and additional regulatory elements (i.e., upstream activating sequences, enhancers and silencers) which alter gene expression in response to developmental and/or external stimuli, or in a tissue-specific manner.
  • promoter is also used to describe a regulatory sequence that confers, activates or enhances the expression of an MCC gene to which it is operably connected and which encodes an MCC polypeptide in vivo.
  • operable connection is meant that the promoter is positioned such that it regulates expression of the MCC gene, generally 5' (upstream) relative to the coding sequence and positioned within 2 kb of the start site of transcription of the gene.
  • the 5 -untranslated region of a human MCC gene i.e. SEQ ID NO: 1
  • SEQ ID NO: 3 the 5 -untranslated region of a human MCC gene
  • the first 220 nucleotides of transcribed sequence in SEQ ID NO: 1 correspond to the 3 '-terminal 220 residues of SEQ ID NO: 3.
  • the nucleic acid that regulates expression of an MCC gene comprises the nucleotide sequence set forth in SEQ ID NO: 3, more preferably, nucleotides from about position 1 to about position 560 of SEQ ID NO: 3 and still more preferably nucleotides from about position 280 to about position 404 of SEQ ID NO: 3.
  • the term "about” shall be taken to include at least 1-10 nucleotide residues upstream or downstream of the stated residue limitations.
  • hyper methylation of several CpG islands in the 5 '-terminal 560 residues of SEQ ID NO: 3 is diagnostic of colorectal cancer, with hyper methylation of a region from position 292 to position 458 has a higher correlation with carcinoma in situ especially the occurrence of hype ⁇ lastic polyps, or stage C colorectal cancer. Accordingly, the detection of hyper methylated residues in an amplified nucleic acid fragment comprising these nucleotides, in particular an amplified fragment consisting of residues 284 to 404, has diagnostic utility in the methods described herein.
  • the detection of hyper methylated residues in a segment selected from the group consisting of: (i) a segment comprising residues from position 284 to position 304; and/or (ii) a segment comprising residues from position 335 to position 355; and/or (iii) a segment comprising residues from position 361 to position 404, is diagnostic of colorectal cancer in a human subject.
  • methylation of nucleic acid shall be taken to mean the addition of a methyl group by the action of a DNA methyl transferase enzyme to a "CpG island" of nucleic acid, eg., genomic DNA.
  • a DNA methyl transferase enzyme e.g., a DNA methyl transferase enzyme
  • hyper methylation of a CpG island results in a "closed” conformation that reduces or silences gene expression.
  • enhanced is meant that there are a significantly larger number of methylated CpG islands in the subject diagnosed than in the reference or suitable control sample.
  • the present invention is not to be limited by a precise number of methylated residues that are considered to be diagnostic of colorectal cancer in a subject, because some variation between patient samples will occur depending upon the tumor stage, positioning of the methylated residue and other global effects such as, for example, a secondary structure of nucleic acid that may hinder detection of methylated residues. For example, notwithstanding extensive methylation in the 5 -untranslated region of a human MCC gene (i.e.
  • SEQ ID NO: 1 in cancer cells lines derived from hype ⁇ lastic polyps or Stage C patients, several CpG sites may be protected directly or indirectly from methylation by secondary structure, or variably methylated between samples. Moreover, notwithstanding the significant utility of methylation within residues 284 to 404 of SEQ ID NO: 3 as a diagnostic, not all subjects having colorectal cancer will necessarily exhibit methylation of all residues in the region consisting of residues 284 to 404 of SEQ ID NO: 3.
  • At least about 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 CpG islands are methylated in a subject having hype ⁇ lastic polyps, serrated adenoma, mixed polyps, hype ⁇ lastic polyposis or more advanced colorectal cancer.
  • colorectal cancer at any stage of the disease, including a stage as early as the transition from Stage 0 (i.e., polyposis) to Stage I (i.e., colorectal tumor localized to the colon lining) or Stage II (i.e., tumor that has penetrated the wall of the colon into the abdominal cavity but not elsewhere).
  • Stage I i.e., colorectal tumor localized to the colon lining
  • Stage II i.e., tumor that has penetrated the wall of the colon into the abdominal cavity but not elsewhere.
  • the detection of colorectal cancer at a later stage of the disease is also encompassed by the present invention, such as, for example, the detection of Stage III or Stage IV colorectal cancers.
  • Such enhanced methylation will preferably be apparent in at least about 10-20% of subjects having Stage O or Stage A or Stage B or Stage C colorectal cancer or colorectal cancer at a later stage of the disease. More preferably, enhanced methylation will preferably be apparent in at least about 20-30% of subjects having colorectal cancer at a stage of carcinoma in situ (especially the occurrence of hype ⁇ lastic polyps, serrated polyps or mixed polyps) to Stage C. More preferably, enhanced methylation will preferably be apparent in at least about 30-40% or 40-50% or more of subjects having colorectal cancer at a stage of carcinoma in situ to Stage C.
  • the enhanced methylation of MCC promoter region is detected in at least about 80% to about 100% of hype ⁇ lastic polyps (HP) and at least about 50% of Stage C colorectal tumors, especially HP and tumors from the proximal colon.
  • HP hype ⁇ lastic polyps
  • Stage C colorectal tumors especially HP and tumors from the proximal colon.
  • the present invention is useful for detecting a polyp or tumor (e.g., HP, SA, MP, or Stage C tumor etc.), elsewhere in the colorectum e.g., the splenic flexure and/or distal colon.
  • the present invention is also useful for detecting and/or diagnosing a cancer/tumor selected from the group consisting of carcinoma, adenocarcinoma, sarcoma, lymphoma, carcinoid stromal tumor and gastrointestinal stromal tumor in a subject at any stage of the tumor.
  • the enhanced methylation in a subject sample is determined using a process comprising treating the nucleic acid with an amount of a methylation- sensitive restriction endonuclease enzyme under conditions sufficient for nucleic acid to be digested and then detecting the fragments produced.
  • a methylation- sensitive restriction endonuclease enzyme include Hpa and Hpall.
  • assays include internal controls that are digested with a methylation- insensitive enzyme having the same specificity as the methylation-sensitive enzyme employed.
  • the methylation-insensitive enzyme Mspl is an isoschizomer of the methylation-sensitive enzyme Hpall.
  • Suitable detection methods for achieving selective hybridisation to the hybridisation probe include, for example, Southern or other nucleic acid hybridization (Kawai et al, Mol Cell. Biol. 14, 7421-7427, 1994; Gonzalgo et al, Cancer Res. 57, 594-599, 1997).
  • the term "selectively hybridizable" means that the probe is used under conditions where a target nucleic acid, e.g., the MCC gene promoter, hybridizes to the probe to produce a signal that is significantly above background (i.e., a high signal-to-noise ratio).
  • the intensity of hybridization is measured, for example, by radiolabeling the probe, e.g. by inco ⁇ orating [ ⁇ - 35 S] and/or [ ⁇ - 32 P]dNTPs, [ ⁇ - 32 P]ATP, biotin, a dye ligand (e.g., FAM or TAMRA), fluorophore, or other suitable ligand into the probe prior to use and then detecting the ligand following hybridization.
  • Suitable hybridization conditions are determined based on the melting temperature (Tm) of a nucleic acid duplex comprising the probe, e.g., as described by Berger and Kimmel (1987, Guide to Molecular Cloning Techniques, Methods in Enzymology, Vol 152, Academic Press, San Diego CA). The skilled artisan will be aware that optimum hybridisation reaction conditions should be determined empirically for each probe, although some generalities can be applied.
  • hybridisations employing short oligonucleotide probes are performed at low to medium stringency. For the pu ⁇ oses of defining the level of stringency, a low- stringency hybridization is achieved using a hybridization buffer and/or a wash solution comprising the following:
  • lxSSC consists of 0.15 M NaCl, 0.015 M Na 3 Citrate pH 7.0.
  • Moderate stringency hybridisation conditions will generally be achieved using a hybridization buffer and/or a wash solution comprising:
  • High stringency hybridisation conditions can achieve a high signal-to- noise ratio as desired.
  • High stringency hybridisation conditions will generally be achieved using a hybridization buffer and/or a wash solution comprising: (i) a salt concentration that is equivalent to about O.lxSSC buffer or lower salt concentration; (ii) a detergent concentration equivalent to 0.1% (w/v) SDS or lower detergent concentration; and (iii) an incubation temperature above about 55°C and preferably above about 65 °C .
  • Conditions for specifically hybridizing nucleic acid, and conditions for washing to remove non-specific hybridizing nucleic acid are well understood by those skilled in the art.
  • any suitable hybridisation probe derived from the promoter region of the MCC gene set forth in SEQ ID NO: 3 can be used in accordance with standard procedures. This is because the detection involves hybridisation to all fragments produced, as opposed to a selective hybridization, and then comparing the fragments produced in the test sample to those fragments produced for a suitable control sample.
  • Preferred hybridization probes will comprise at least about 18 contiguous nucleotides in length from SEQ ID NO: 3, more preferably at least about 50 contiguous nucleotides from SEQ ID NO: 3, preferably inco ⁇ orating one or more CpG islands that are hyper methylated in colorectal cancer.
  • Preferred probes will hybridise to a region that includes residues 284-404 of SEQ ID NO: 3 or a sequence that is complementary thereto, or a portion thereof including one or more CpG islands that are hyper methylated in colorectal cancer.
  • the probe will include the Mspl/Hpall recognition site located at position 291 to position 294 of SEQ ID NO: 3.
  • digestion of nucleic acid from a colorectal cancer sample with Hpall will fail to cleave at this position, thereby producing a larger DNA fragment than for a healthy/normal control sample in which the site is not methylated.
  • the methylation-insensitive enzyme Mspl will produce the same-size fragment for both the colorectal cancer sample and the control sample.
  • longer probes are preferred, because these generally produce higher signal-to-noise ratio than shorter probes and/or permit higher stringency hybridisation and wash conditions to be employed.
  • hybridization probes that comprise at least about 100 contiguous nucleotides from SEQ ID NO: 3 and even more preferably at least about 200 contiguous nucleotide residues.
  • restriction fragments derived from the MCC gene promoter are particularly useful for such applications.
  • a difference in the fragments produced for the test sample and a negative control sample is indicative of the subject having colorectal cancer.
  • the control sample comprises data from a tumor, cancer tissue or a cancerous cell or pre-cancerous cell
  • similarity, albeit not necessarily absolute identity between the test sample and the control sample is indicative of a positive diagnosis (i.e. colorectal cancer).
  • Methylation-sensitive endonuclease-based assays are straightforward, however are generally of low sensitivity and require large amounts of input DNA, unless linked to a PCR detection method. Such linkage is clearly encompassed by the present invention.
  • the fragments produced by the restriction enzyme are detected using an amplification system, such as, for example, polymerase chain reaction (PCR), rolling circle amplification (RCA), inverse polymerase chain reaction (iPCR), in situ PCR (Singer-Sam et al, Nucl Acids Res. 18, 687,1990), strand displacement amplification (SDA) or cycling probe technology.
  • amplification system such as, for example, polymerase chain reaction (PCR), rolling circle amplification (RCA), inverse polymerase chain reaction (iPCR), in situ PCR (Singer-Sam et al, Nucl Acids Res. 18, 687,1990), strand displacement amplification (SDA) or cycling probe technology.
  • nucleic acid primer molecules comprising at least about 18 nucleotides in length, and more preferably at least 20-30 nucleotides in length are hybridised to different strands of a nucleic acid template molecule at their respective annealing sites, and specific nucleic acid molecule copies of the template that intervene the annealing sites are amplified enzymatically.
  • Amplification products may be detected using electrophoresis and detection with a detectable marker that binds nucleic acids.
  • one or more of the oligonucleotides are labelled with a detectable marker (e.g. a fluorophore) and the amplification product detected using, for example, a lightcycler (Perkin Elmer, Wellesley, MA, USA).
  • a detectable marker e.g. a fluorophore
  • a lightcycler Perkin Elmer, Wellesley, MA, USA.
  • Strand displacement amplification utilises oligonucleotide primers, a DNA polymerase and a restriction endonuclease to amplify a target sequence.
  • the oligonucleotides are hybridized to a target nucleic acid and the polymerase is used to produce a copy of the region intervening the primer annealing sites.
  • the duplexes of copied nucleic acid and target nucleic acid are then nicked with an endonuclease that specifically recognises a sequence at the beginning of the copied nucleic acid.
  • the DNA polymerase recognises the nicked DNA and produces another copy of the target region at the same time displacing the previously generated nucleic acid.
  • SDA Strand displacement amplification
  • RNA-DNA duplex formed is a target for RNaseH thereby cleaving the primer. The cleaved primer is then detected.
  • Preferred amplification primers will comprise at least about 18 contiguous nucleotides in length from SEQ ID NO: 3, more preferably at least about 50 contiguous nucleotides from SEQ ID NO: 3, preferably inco ⁇ orating one or more CpG islands that are hyper methylated in colorectal cancer and flanking or comprising a methylation-sensitive endonuclease recognition site.
  • primers that flank a methylation-sensitive endonuclease recognition site it is preferred that such primers flank only those sites that are hyper methylated in colorectal cancer to ensure that a diagnostic amplification product is produced.
  • Preferred primers will flank a region that includes residues 284-404 of SEQ ID NO: 3 or a sequence that is complementary thereto, or a portion thereof including one or more CpG islands that are hyper methylated in colorectal cancer.
  • the amplification primers are selected such that they flank the Mspll Hpall recognition site located at position 291 to position 294 of SEQ ID NO: 3.
  • amplified nucleic acid from a healthy control but not from a colorectal cancer sample will cleave at this position following digestion with Hpall.
  • Amplification products using such primers are expected for both a colorectal cancer sample and a normal/healthy control, however a larger amplification product is produced for a colorectal cancer sample than for a control sample as a consequence of cleavage at the Hpall recognition site by the enzyme Hpall.
  • the shorter amplification product is produced for both a colorectal cancer sample and a normal/healthy control using such primers.
  • the precise length of the amplified product will vary depending upon the distance between the primers.
  • At least one amplification primer will comprise the Mspl/Hpall recognition site located at position 291 to position 294 of SEQ ID NO: 3 or a sequence that is capable of hybridising to said recognition site or the complement thereof. Accordingly, a primer that comprises this recognition sequence will produce an amplification product only when the site is methylated. In contrast, the me hylation- insensitive enzyme Mspl will produce the same-size fragment for both the colorectal cancer sample and the control sample.
  • the primer includes the Hpall recognition sequence at its 3 '-end such that amplification of downstream nucleic acid occurs when the Hpall recognition sequence has not been cleaved (i.e., in the colorectal cancer sample), and not when cleaved (i.e., in a healthy or normal control sample) by Hpall.
  • a negative control amplification reaction employing Mspl no amplification product is produced using such a primer.
  • one or more of the primers may be labelled with a detectable marker to facilitate rapid detection of amplified nucleic acid, for example, a fluorescent label (e.g. Cy5 or Cy3) or a radioisotope (e.g. P).
  • a detectable marker for example, a fluorescent label (e.g. Cy5 or Cy3) or a radioisotope (e.g. P).
  • the amplified nucleic acids are generally analysed using, for example, non-denaturing polyacrylamide gel electrophoresis, mass spectrometry (e.g., MALDI-TOF, Sequenome), liquid chromatography (eg. ⁇ PLC or d ⁇ PLC), or capillary electrophoresis.
  • mass spectrometry e.g., MALDI-TOF, Sequenome
  • liquid chromatography eg. ⁇ PLC or d ⁇ PLC
  • capillary electrophoresis capillary electrophoresis.
  • High throughput detection methods such as, for example, matrix- assisted laser deso ⁇ tion/ionization time of flight (MALDI-TOF), electrospray ionization (ESI), Mass spectrometry (including tandem mass spectrometry, eg LC MS/MS), biosensor technology, evanescent fiber-optics technology or DNA chip technology (e.g., WO98/49557; WO 96/17958; Fodor et al, Science 161-113, 1991; U.S. Pat. No. 5,143,854; and U.S. Patent No.
  • MALDI-TOF matrix- assisted laser deso ⁇ tion/ionization time of flight
  • ESI electrospray ionization
  • Mass spectrometry including tandem mass spectrometry, eg LC MS/MS
  • biosensor technology evanescent fiber-optics technology or DNA chip technology
  • DNA chip technology e.g., WO98/49557; WO 96/17958; Fodor et
  • 5,837,832 the contents of which are all inco ⁇ orated herein by reference), are especially preferred for all assay formats described herein, especially when screening of large numbers of samples, such as, for example, public health screening of subjects having a higher risk of developing colorectal cancer.
  • a particular example of a suitable solid substrate for producing DNA chip arrays is the commercially available BIACoreTM chip (Pharmacia Biosensors).
  • amplification of a nucleic acid may be continuously monitored using a melting curve analysis method, such as that described in, for example, US 6,174,670, which is inco ⁇ orated herein by reference.
  • a melting curve analysis method such as that described in, for example, US 6,174,670, which is inco ⁇ orated herein by reference.
  • the nucleotide sequence of the amplified DNA is determined according to standard procedures.
  • the enhanced methylation in a subject sample is determined by performing a process comprising treating the nucleic acid with an amount of DNasel under conditions sufficient for nucleic acid to be digested and then detecting the fragments produced.
  • This assay format is predicated on the understanding that methylated DNA has a more tightly-closed conformation than non-hyper methylated DNA and, as a consequence, is less susceptible to endonuclease digestion by DNase I.
  • DNA fragments of different lengths are produced by DNase I digestion of methylated compared to non-hyper methylated DNA.
  • art- recognized detection methods are employed, such as, for example, Southern or other nucleic acid hybridization (Kawai et al, Mol. Cell. Biol. 14, 7421-7427, 1994; Gonzalgo et al, Cancer Res. 57, 594-599, 1997).
  • assay formats are generally described herein above and apply mutatis mutandis to SSCP -based processes presently described.
  • the probe can be selected from any portion of SEQ ID NO: 3 that inco ⁇ orates or flanks a region that is hyper methylated in colorectal cancer.
  • Preferred probes will hybridise to a region that includes residues 284-404 of SEQ ID NO: 3 or a sequence that is complementary thereto, or a portion thereof.
  • a difference in the length of fragments produced for the test sample and a negative control sample is indicative of the subject having colorectal cancer.
  • a larger fragment is produced from the colorectal cancer patient sample.
  • the control sample comprises data from a tumor, cancer tissue or a cancerous cell or pre-cancerous cell
  • similarity, albeit not necessarily absolute identity, between the test sample and the control sample is indicative of a positive diagnosis (i.e. colorectal cancer).
  • High throughput detection methods such as, for example, matrix-assisted laser deso ⁇ tion/ionization time of flight (MALDI-TOF), electrospray ionization (ESI), Mass spectrometry (including tandem mass spectrometry, eg LC MS/MS), biosensor technology, evanescent fiber-optics technology or DNA chip technology, can also be employed.
  • MALDI-TOF matrix-assisted laser deso ⁇ tion/ionization time of flight
  • ESI electrospray ionization
  • Mass spectrometry including tandem mass spectrometry, eg LC MS/MS
  • biosensor technology evanescent fiber-optics technology or DNA chip technology
  • an amplification assay is used to detect the fragments of differing length produced by DNase I digestion, such as, for example, polymerase chain reaction (PCR), rolling circle amplification (RCA), inverse polymerase chain reaction (iPCR), in situ PCR (Singer-Sam et al, Nucl. Acids Res. 18, 687,1990), strand displacement amplification, or cycling probe technology.
  • PCR polymerase chain reaction
  • RCA rolling circle amplification
  • iPCR inverse polymerase chain reaction
  • in situ PCR Singer-Sam et al, Nucl. Acids Res. 18, 687,1990
  • strand displacement amplification or cycling probe technology.
  • PCR-SSCP is performed essentially as described by Gregory and Feil Nucleic Acids Res., 27, e32i-e32iv, 1999.
  • High throughput detection methods such as, for example, matrix-assisted laser deso ⁇ tion/ionization time of flight (MALDI-TOF), electrospray ionization (ESI), Mass spectrometry (including tandem mass spectrometry, eg LC MS/MS), biosensor technology, evanescent fiber-optics technology or DNA chip technology, can also be employed.
  • the assay format comprises a positive read-out system in which DNA from a colorectal cancer sample is detected as a positive signal.
  • nucleic acid is subjected to DNase I digestion and the fragments produced are amplified using primers that bind to or flank methylated regions of the MCC gene promoter in a polymerase chain reaction or equivalent detection means.
  • amplification primers flanking or comprising one or more CpG islands that are resistant to DNase I digestion in a colorectal cancer sample but not resistant to DNase I digestion in a healthy/normal control or healthy/normal test sample are used to amplify the DNase I-generated fragments.
  • the production of a specific nucleic acid fragment using DNase I is diagnostic of colorectal cancer, because the DNA is not efficiently degraded.
  • template DNA from a healthy/normal subject sample is degraded by the action of DNase I and, as a consequence, amplification fails to produce a discrete amplification product.
  • the primers are selected from the group consisting of (i) a primer comprising a sequence contained within residues from position 284 to position 304 of SEQ ID NO: 3; (ii) a primer comprising a sequence contained within residues from position 335 to position 355 of SEQ ID NO: 3; (iii) a primer comprising a sequence contained within residues from position 361 to position 382 of SEQ ID NO: 3; (iv) a primer comprising a sequence contained within residues from position 383 to position 404 of SEQ ID NO: 3; and (v) a primer comprising a sequence that is complementary to any one of (i) to (iv).
  • each primer will comprise a sequence as set forth in SEQ ID Nos: 11-16 or complementary to any one or more of SEQ ID Nos: 11-16. Minor sequence variants thereof that have substantially the same melting temperature as the exemplified probes may also be used.
  • the nucleotide sequence set forth in SEQ ID NO: 11 corresponds to residues at positions 284 to 304 of SEQ ID NO: 3 in the 5 -untranslated region of the MCC gene promoter.
  • the sequence set forth in SEQ ID NO: 12 corresponds to residues at positions 335 to 355 of SEQ ID NO: 3 in the 5 -untranslated region of the MCC gene promoter.
  • sequence set forth in SEQ ID NO: 13 corresponds to the complement of residues at positions 361 to 382 of SEQ ID NO: 3 in the 5 -untranslated region of the MCC gene promoter.
  • sequence set forth in SEQ ID NO: 14 corresponds to residues at positions 335 to 354 of SEQ ID NO: 3 in the 5 - untranslated region of the MCC gene promoter.
  • sequence set forth in SEQ ID NO: 14 corresponds to residues at positions 335 to 354 of SEQ ID NO: 3 in the 5 - untranslated region of the MCC gene promoter.
  • 16 corresponds to the complement of residues at positions 383 to 404 of SEQ ID NO: 3 in the 5 -untranslated region of the MCC gene promoter.
  • the primers used will be a combination selected from the group consisting of:
  • the detection step of the assay formats described herein clearly encompass the use of multiple rounds of amplifications and/or combinations of amplification and classical nucleic acid hybridization steps, in any order.
  • nucleic acid of the MCC gene promoter using a combination of a primer comprising the sequence set forth in SEQ ID NO: 14 and a primer comprising the sequence of SEQ ID NO: 16, and then to hybridize the amplified nucleic acid using a labeled hybridization probe that comprises the nucleotide sequence of SEQ ID NO: 15 or a complementary sequence thereto.
  • a labeled hybridization probe that comprises the nucleotide sequence of SEQ ID NO: 15 or a complementary sequence thereto.
  • hybridization probes capable of hybridizing to the amplified nucleic acid is clearly contemplated by the present invention, the only requirement being that the probe comprises a nucleotide sequence that occurs within the MCC gene promoter at a position between the two amplification primer sequences and preferably does not comprise a sequence of either amplification primer used.
  • a first series of amplification reactions may employ a primer comprising the sequence set forth in SEQ ID NO: 11 and a primer comprising the sequence of SEQ ID NO: 13, and a second series of amplification reactions may employ: (i) a primer comprising the sequence set forth in SEQ ID NO: 11 and a primer comprising a sequence that is complementary to the sequence of SEQ ID NO: 12; or alternatively, (ii) a primer comprising the sequence set forth in SEQ ID NO: 12 and a primer comprising the sequence of SEQ ID NO: 13; or alternatively, (iii) a primer comprising the sequence set forth in SEQ ID NO: 12 and a primer comprising the sequence of SEQ ID NO: 14; or alternatively, (iv) a primer comprising the sequence of SEQ ID NO: 12 and a primer comprising the a sequence that is complementary to SEQ ID
  • a second series of amplification reactions may employ any one or more of the sequences set forth in SEQ ID Nos: 12-15 in an appropriate combination as described supra to amplify the intervening region there between.
  • the performance of each and every of the above-mentioned second series of amplification reactions simultaneously or contemporaneously is also encompassed by the present invention.
  • primer combinations are also not to be excluded when using multiple amplifications to detect nucleic acid, the only requirement being that the primers are selected such that they comprise nucleotide sequences that occur within the MCC gene promoter at a position between the two amplification primer sequences used for the first series of amplifications.
  • the skilled artisan will readily be capable of determining the nucleotide sequence of suitable amplification primers to perform this embodiment based upon the disclosure in SEQ ID NO: 3 and, as a consequence, the present invention is not to be limited by the precise sequence of second round amplification primers used in conjunction with the first round amplification primers as described.
  • Combinations of the exemplified primers set forth in SEQ ID Nos: 11-16 are preferred for first and second round amplifications because these are derived from methylated sequences of the MCC gene promoter and, as a consequence, provide enhanced specificity in the detection of hyper methylated sequences.
  • methylated sequence-specific primers in all detection stages also provides for validation of a primary diagnosis of colorectal cancer based on the use of a single primer set or hybridization probe, and a reduced level of false positive diagnoses than would otherwise be the case.
  • methylation of cytosine residues within the CpG islands of these sequences in the MCC gene promoter is enhanced and, as a consequence, protected from DNase I digestion. This enables enhanced hybridization of one or more probes to the MCC gene promoter in the cancer sample thereby producing an enhanced signal relative to a normal/healthy control.
  • the present invention also encompasses the use of real-time quantitative forms of PCR, such as, for example, TaqMan (Holland et al, Proc. Natl Acad. Sci. USA, 88, 1216- 7280, 1991; Lee et al, Nucleic Acid Res. 21, 3161-3166, 1993) to perform this embodiment.
  • TaqMan indicates the probe used to detect specific sequences in amplified PCR products by employing the 5 - to 3 -exonuclease activity of Tag DNA polymerase.
  • a TaqMan probe (about 20-30 nucleotides in length), disabled from extension at the 3 - end, generally consists of a site-specific sequence labeled with a fluorescent reporter dye and a fluorescent quencher dye e.g., TAMRA or FAM, or alternatively or in addition, a Black Hole Quencher (BHQ) fluorescent dye that prevents fluorescence until a hybridization event occurs (Biosearch Technologies, Inc., Novato CA 94949-5750, USA).
  • a fluorescent reporter dye e.g., TAMRA or FAM
  • BHQ Black Hole Quencher
  • each of the fluorescent reporter dye, fluorescent quencher dye and BHQ may be added to the 5 -end or 3 -end of the probe and the present invneiton encompasses all such alternatives.
  • the TaqMan probe hybridizes to its complementary single strand DNA sequence within the methylated region of the MCC gene promoter.
  • the TaqMan probe is degraded due to the 5 ⁇ >3' exonuclease activity of Tag DNA polymerase, thereby separating the quencher from the reporter during extension. Due to the release of the quenching effect on the reporter, the fluorescence intensity of the reporter dye increases. During the entire amplification process this light emission increases exponentially, the final level being measured by spectrophotometry upon termination of the PCR.
  • the TaqMan assay offers a sensitive method to determine the presence or absence of specific sequences.
  • the TaqMan assay allows high sample throughput because no gel-electrophoresis is required for detection.
  • TaqMan probe comprising the sequences set forth in SEQ ID NOs: 12 or 15 labelled at its 5'- and 3'- ends with different fluorescent ligands, e.g., TAMRA and/or FAM and/or BHQ.
  • the probe is labelled at the 5 '-end using FAM and BHQ and at the 3 '-end using TAMRA, or alternatively, at the 5 -end with TAMRA and BHQ and at the 3 -end with FAM, or alternatively, at the 5 '-end using FAM and at the 3 '-end using TAMRA and BHQ, or alternatively, at the 5 -end with TAMRA and at the 3 -end with FAM and BHQ.
  • Such a probe is generally used in conjunction with amplification primers having different and non-overlapping sequences with SEQ ID NOs: 12 or 15 in the MCC gene promoter, as described herein above.
  • the amplification primers set forth in SEQ ID Nos: 11 and 13 are used in conjunction with a TaqMan probe comprising the nucleotide sequence of SEQ ID NO: 12.
  • the amplification primers set forth in SEQ ID Nos: 14 and 16 are used in conjunction with a TaqMan probe comprising the nucleotide sequence of SEQ ID NO: 15.
  • the enhanced methylation in a subject sample is determined using a process comprising treating the nucleic acid with an amount of a compound that selectively mutates a non-methylated cytosine residue within a CpG island under conditions sufficient to induce mutagenesis.
  • Preferred compounds mutate cytosine to uracil or thymidine, such as, for example, a metal salt of bisulphite, eg., sodium bisulphite or potassium bisulphite (Frommer et al, Proc. Natl Acad. Sci. USA 89, 1827-1831, 1992).
  • Bisulfite treatment of DNA is known to distinguish methylated from non-methylated cytosine residues, by mutating cytosine residues that are not protected by methylation, including cytosine residues that are not within a CpG island or that are positioned within a CpG island that is not subject to methylation. Both strands of a sequence are susceptible to mutagenesis using bisulphite.
  • SEQ ID NO: 17 The nucleotide sequence of one strand of bisulphite treated DNA, containing the methylated region of the MCC gene promoter is set forth in SEQ ID NO: 17.
  • the nucleotide sequence set forth in SEQ ID NO: 17 relates to the 5 -untranslated region of a bisulphite-mutated MCC gene comprising cytosine residues that have been protected from mutagenesis by hyper methylation in colorectal cancer cells.
  • SEQ ID NO: 17 provides a mutated version of the wild- type sequence set forth in SEQ ID NO: 3.
  • SEQ ID NO: 24 The nucleotide sequence of a non-hyper methylated MCC gene promoter following bisulphite treatment is set forth in SEQ ID NO: 24.
  • the nucleotide sequence set forth in SEQ ID NO: 24 relates to the 5 -untranslated region of a bisulphite-mutated MCC gene wherein, due to the absence of methylation, diagnostic cytosine residues have not been protected from mutagenesis.
  • SEQ ID NO: 24 provides a mutated version of the wild-type sequence set forth in SEQ ID NO: 3 and differs from SEQ ID NO: 17 in so far as diagnostic CpG islands have not been protected from mutation.
  • nucleotide sequences of both the methylated and non-methylated forms of the MCC promoter may readily determine nucleotide sequences of both the methylated and non-methylated forms of the MCC promoter, by performing bisulphite sequencing of the complementary strands in normal and colorectal cancer tissues. This generally involves determining the pattern of methylation on the opposing strand and/or the nucleotide sequences of the opposing strands to SEQ ID Nos: 3, 17 or 24 following bisulphite treatment. In this respect, the cytosine residues on both strands are converted to thymine residues by bisulphite unless protected by methylation.
  • probes and primers for performing the following embodiments of the invention are readily derived from SEQ ID NO: 17 and/or SEQ ID NO: 24.
  • the assay format comprises a positive read-out system in which DNA from a colorectal cancer sample is detected as a positive signal.
  • the non-hyper methylated DNA from a healthy or normal control subject is not detected or only weakly detected.
  • the enhanced methylation in a subject sample is determined using a process comprising: (i) treating the nucleic acid with an amount of a compound that selectively mutates a non-methylated cytosine residue within a CpG island under conditions sufficient to induce mutagenesis thereby producing a mutated nucleic acid; (ii) hybridizing the nucleic acid to a probe or primer comprising a nucleotide sequence that is complementary to a sequence comprising the methylated cytosine residue under conditions such that selective hybridization to the non- mutated nucleic acid occurs; and (iii) detecting the selective hybridization.
  • the term "selective hybridisation” means that hybridisation of a probe or primer to the non-mutated nucleic acid occurs at a higher frequency or rate, or has a higher maximum reaction velocity, than hybridisation of the same probe or primer to the corresponding mutated sequence.
  • the probe or primer does not hybridise to the non-hyper methylated sequence carrying the mutation(s) under the reaction conditions used.
  • the hybridisation is detected using Southern, dot blot, slot blot or other nucleic acid hybridisation means (Kawai et al, Mol. Cell. Biol. 14, 7421-7427, 1994; Gonzalgo et al, Cancer Res. 57, 594-599, 1997).
  • Subject to appropriate probe selection, such assay formats are generally described herein above and apply mutatis mutandis to the presently described selective mutagenesis approach.
  • a ligase chain reaction format is employed to distinguish between a mutated and non-mutated MCC gene promoter.
  • Ligase chain reaction (described in EP 320,308 and US 4,883,750) uses at least two oligonucleotide probes that anneal to a target nucleic acid in such a way that they are juxtaposed on the target nucleic acid (i.e., MCC gene promoter sequence).
  • the target nucleic acid is hybridised to a first probe that is complementary to a diagnostic portion of the target sequence (the diagnostic probe) e.g., a methylated CpG island, and with a second probe that is complementary to a nucleotide sequence contiguous with the diagnostic portion (the contiguous probe), under conditions wherein the diagnostic probe remains bound substantially only to the target nucleic acid.
  • the diagnostic probe e.g., a methylated CpG island
  • the diagnostic and contiguous probes can be of different lengths and/or have different melting temperatures such that the stringency of the hybridization can be adjusted to permit their selective hybridisation to the target, wherein the probe having the higher melting temperature is hybridised at higher stringency and, following washing to remove unbound and/or non-selectively bound probe, the other probe having the lower melting temperature is hybridised at lower stringency.
  • the diagnostic probe and contiguous probe are then covalently ligated such as, for example, using T4 DNA ligase, to thereby produce a larger target probe that is complementary to the target sequence, and the probes that are not ligated are removed by modifying the hybridisation stringency.
  • the stringency of the hybridisation can be increased to a stringency that is at least as high as the stringency used to hybridise the longer probe, and preferably at a higher stringency due to the increased length contributed by the shorter probe following ligation.
  • the target-probe duplex it is preferred to melt the target-probe duplex, elute the dissociated probe and confirm that is has been ligated, e.g., by determining its length using electrophoresis, mass spectrometry, nucleotide sequence analysis, gel filtration, or other means known to the skilled artisan.
  • ligase chain reaction is particularly sensitive for distinguishing between multiple methylated residues and multiple non-hyper methylated residues, by careful selection of combinations of probes based upon the sequence set forth in SEQ ID NO: 17. Accordingly, in an alternative embodiment of the ligase chain reaction, the ligated probes are detected by adding an "anchor" primer following the ligation reaction and performing PCR in accordance with standard amplification procedures as described herein.
  • the “anchor" primer is selected such that it comprises a nucleotide sequence that will anneal to the opposing strand to the ligated probe and have a 5'- to 3'- orientation that will permit amplification of target nucleic acid that intervenes the ligated probe and anchor primer.
  • the length of the amplification product can then be confirmed in the usual manner wherein a longer product confirms the ligation event.
  • one or both of the probes is labeled such that the presence or absence of the target sequence can be tested by melting the target-probe duplex, eluting the dissociated probe, and testing for the label(s).
  • both probes are labelled, different ligands are used to permit distinction between the ligated and unligated probes, in which case the presence of both labels in the same eluate fraction confirms the ligation event.
  • both the diagnostic probe and the contiguous probe are required to anneal to their respective target sites in the target nucleic acid for the label to appear in the assay.
  • the target nucleic acid is bound to a solid matrix e.g., in a Southern hybridisation, slot blot, dot blot, or microchip assay format, the presence of both the diagnostic and contiguous probes can be determined directly.
  • a solid matrix e.g., in a Southern hybridisation, slot blot, dot blot, or microchip assay format
  • Probes suitable for such an assay format are readily derived from the description herein of hyper methylated CpG islands in the 5 -terminal 560 residues of SEQ ID NO: 17.
  • the diagnostic probe and preferably also the contiguous probe should be selected such that they selectively hybridize to wild type sequences in the MCC gene promoter that are methylated in samples from subjects having colorectal cancer and thereby protected from mutation.
  • selectively hybridize in this context is meant that the probe(s) anneal at a significantly higher frequency under the conditions employed to a mutated target sequence of a hyper methylated MCC gene promoter derived from a colorectal cancer sample compared to a mutated target sequence of an MCC gene promoter derived from a healthy or normal control subject, thereby producing a high signal-to-noise ratio in the assay.
  • the probe(s) have 3 '-terminal and/or 5 '-terminal sequences that comprise a CpG island that is hyper methylated in colorectal cancer compared to a healthy or normal control subject, such that the diagnostic probe and contiguous probe are capable of being ligated only when the cytosine of the CpG island has not been mutated to thymidine e.g., in the case of a methylated cytosine residue.
  • the diagnostic and contiguous probes are selected from the group consisting of (i) a probe comprising a sequence contained within the sequence set forth in SEQ ID NO: 17 and having a 5 '-terminal guanosine residue at a position within SEQ ID NO: 17 selected from the group consisting of position 293, position
  • a probe comprising a sequence contained within the sequence set forth in SEQ ID NO: 17 and having a 3 '-terminal cytosine residue at a position within SEQ ID NO: 17 selected from the group consisting of position 292, position 297, position 302, position 338, position
  • a probe comprising a sequence that is complementary to a portion of SEQ ID NO: 17 and having a 5'-terminal guanosine residue complementary to a cytosine residue within SEQ ID NO: 17 at a position selected from the group consisting of position 292, position 297, position 302, position
  • a probe comprising a sequence that is complementary to a portion of SEQ ID NO: 17 and having a 3 '-terminal cytosine residue complementary to a guanosine residue within SEQ ID NO: 17 at a position selected from the group consisting of position 293, position 298, position 303, position
  • a probe comprising a sequence that is complementary to any one of (i) to (iv).
  • exemplary combinations of diagnostic and contiguous probes that are ligatable are selected by combination of a probe set forth in sub-paragraph (i) supra with a probe set forth in sub-paragraph (ii) supra, or alternatively, by combination of a probe set forth in sub-paragraph (iii) supra with a probe set forth in sub-paragraph (iv).
  • multiple sets of diagnostic probes and multiple sets of contiguous probes can be employed to provide for assay products that detect clusters of methylated residues in the MCC gene promoter. Again, the selection of these probes can be based upon the criteria for probe design described in the preceding paragraphs.
  • the hybridization is detected using an amplification system.
  • amplification system In methylation-specific PCR formats (MSP; Herman et al. Proc. Natl. Acad. Sci. USA 93, 9821-9826, 1992), the hybridization is detection using a process comprising amplifying the bisulphite-treated DNA.
  • MSP methylation-specific PCR formats
  • positive read-out formats methylation of cytosine residues within the CpG islands of these sequences in the MCC gene promoter of a colorectal cancer sample is enhanced and, as a consequence, protected from mutation. This enables enhanced annealing of one or more primers as described to the MCC gene promoter in the cancer sample thereby producing an enhanced signal relative to a normal/healthy control.
  • amplification assay format described herein can be used, such as, for example, polymerase chain reaction (PCR), rolling circle amplification (RCA), inverse polymerase chain reaction (iPCR), in situ PCR (Singer-Sam et al, Nucl. Acids Res. 18, 687,1990), strand displacement amplification, or cycling probe technology.
  • PCR polymerase chain reaction
  • RCA rolling circle amplification
  • iPCR inverse polymerase chain reaction
  • in situ PCR Singer-Sam et al, Nucl. Acids Res. 18, 687,1990
  • strand displacement amplification or cycling probe technology.
  • the primers are selected from the group consisting of (i) a primer comprising a sequence contained within residues from position 284 to position 304 of SEQ ID NO: 17; (ii) a primer comprising a sequence contained within residues from position 335 to position 355 of SEQ ID NO: 17; (iii) a primer comprising a sequence contained within residues from position 361 to position 382 of SEQ ID NO: 17; (v) a primer comprising a sequence contained within residues from position 383 to about position 403 or about position 404 of SEQ ID NO: 17; and (v) a primer comprising a sequence that is complementary to any one of (i) to (iv).
  • Amplification primers will preferably comprise sequences corresponding or complementary to any one or more of SEQ ID Nos: 18-23.
  • the nucleotide sequence set forth in SEQ ID NO: 18 corresponds to residues at positions 284 to 304 of SEQ ID NO: 17 (i.e., in the 5 -untranslated region of the MCC gene promoter from a colorectal cancer sample following bisulphite treatment of DNA).
  • the sequence set forth in SEQ ID NO: 19 corresponds to residues at positions 335 to 355 of SEQ ID NO: 17.
  • sequence set forth in SEQ ID NO: 20 corresponds to the complement of residues at positions 361 to 382 of SEQ ID NO: 17 in the 5 -untranslated region of the MCC gene promoter.
  • sequence set forth in SEQ ID NO: 21 corresponds to residues at positions 335 to 354 of SEQ ID NO: 17.
  • sequence set forth in SEQ ID NO: 22 corresponds to residues at positions 359 to 377 of SEQ ID NO: 17.
  • the sequence set forth in SEQ ID NO: 23 corresponds to the complement of residues at positions 383 to about position 403 or about position 404 of SEQ ID NO: 17.
  • the primers used will be a combination selected from the group consisting of: (i) a primer comprising the sequence set forth in SEQ ID NO: 18 and a primer comprising a sequence that is the complement of SEQ ID NO: 19; (ii) a primer comprising the sequence set forth in SEQ ID NO: 18 and a primer comprising the sequence set forth in SEQ ID NO: 20;
  • a primer comprising the sequence set forth in SEQ ID NO: 22 and a primer comprising the sequence set forth in SEQ ID NO: 23 are also encompassed, and these will be readily identified by the skilled artisan.
  • the detection step of the assay formats described herein clearly encompass the use of multiple rounds of amplifications and/or combinations of amplification and classical nucleic acid hybridization steps, in any order.
  • nucleic acid of the mutated MCC gene promoter using a combination of a primer comprising the sequence set forth in SEQ ID NO: 21 and a primer comprising the sequence of SEQ ID NO: 23, and then to hybridize the amplified nucleic acid using a labeled hybridization probe that comprises the nucleotide sequence of SEQ ID NO: 22 or a complementary sequence thereto.
  • hybridization probes capable of hybridizing to the amplified nucleic acid
  • the probe comprises a nucleotide sequence that occurs within the MCC gene promoter at a position between the two amplification primer sequences and preferably does not comprise a sequence of either amplification primer used.
  • the skilled artisan will readily be capable of determining the nucleotide sequence of a suitable hybridization probe to perform this embodiment based upon the disclosure in SEQ ID NO: 17 and, as a consequence, the present invention is not to be limited by the precise sequence of a hybridization probe used in conjunction with amplification primers as described.
  • a first series of amplification reactions may employ a primer comprising the sequence set forth in SEQ ID NO: 18 and a primer comprising the sequence of SEQ ID NO: 20
  • a second series of amplification reactions may employ: (i) a primer comprising the sequence set forth in SEQ ID NO: 18 and a primer comprising a sequence that is complementary to the sequence of SEQ ID NO: 19; or alternatively, (ii) a primer comprising the sequence set forth in SEQ ID NO: 19 and a primer comprising the sequence of SEQ ID NO: 20; or alternatively, (iii) a primer comprising the sequence set forth in SEQ ID NO: 19 and a primer comprising the sequence of SEQ ID NO: 21; or alternatively, (iv) a primer comprising the sequence of SEQ ID NO: 19 and a primer comprising the a sequence that is complementary to SEQ ID
  • a second series of amplification reactions may employ any one or more of the sequences set forth in SEQ ID Nos: 19-22 in an appropriate combination as described supra to amplify the intervening region there between.
  • the performance of each and every of the above-mentioned second series of amplification reactions simultaneously or contemporaneously is also encompassed by the present invention.
  • primer combinations are also not to be excluded when using multiple amplifications to detect nucleic acid, the only requirement being that the primers are selected such that they comprise nucleotide sequences that occur within the MCC gene promoter at a position between the two amplification primer sequences used for the first series of amplifications.
  • the skilled artisan will readily be capable of determining the nucleotide sequence of suitable amplification primers to perform this embodiment based upon the disclosure in SEQ ID NO: 17 and, as a consequence, the present invention is not to be limited by the precise sequence of second round amplification primers used in conjunction with the first round amplification primers as described.
  • Combinations of the exemplified primers set forth in SEQ ID Nos: 18-23 are preferred for first and second round amplifications because these are derived from methylated sequences of the MCC gene promoter and, as a consequence, provide enhanced specificity in the detection of hyper methylated sequences.
  • the use of such methylated sequence-specific primers in all detection stages also provides for validation of a primary diagnosis of colorectal cancer based on the use of a single primer set or hybridization probe, and a reduced level of false positive diagnoses than would otherwise be the case.
  • the present invention also encompasses the use of real-time quantitative forms of PCR, such as, for example, TaqMan (Holland et al, Proc. Natl Acad. Sci. USA, 88, 7276- 7280, 1991; Lee et al, Nucleic Acid Res. 21, 3761-3766, 1993) to perform this embodiment.
  • TaqMan Holland et al, Proc. Natl Acad. Sci. USA, 88, 7276- 7280, 1991; Lee et al, Nucleic Acid Res. 21, 3761-3766, 1993
  • a TaqMan probe comprising the sequence set forth in SEQ ID NOs: 19 or 22 or complementary thereto labelled at its 5'- and 3'- ends with different fluorescent ligands, e.g., TAMRA and/or FAM and/or a BHQ.
  • the probe is labelled at the 5'-end using FAM and at the 3 '-end using TAMRA.
  • amplification primers having different and non-overlapping sequences with SEQ ID NOs: 19 or 22 in the MCC gene promoter, as described herein above.
  • the amplification primers set forth in SEQ ID Nos: 18 and 20 are used in conjunction with a TaqMan probe comprising the nucleotide sequence of SEQ ID NO: 19.
  • the amplification primers set forth in SEQ ID Nos: 21 and 23 are used in conjunction with a TaqMan probe comprising the nucleotide sequence of SEQ ID NO: 22.
  • the amplification product is analysed using a range of procedures, including gel electrophoresis, gel filtration, mass spectrometry, and in the case of labeled primers, by identifying the label in the amplification product.
  • restriction enzyme digestion of PCR products amplified from bisulfite-converted DNA is performed essentially as described by Sadri and Hornsby, Nucl. Acids Res. 24, 5058-5059, 1996; and Xiong and Laird, Nucl. Acids Res. 25, 2532-2534, 1997), to analyze the product formed.
  • High throughput detection methods such as, for example, matrix-assisted laser deso ⁇ tion/ionization time of flight (MALDI-TOF), electrospray ionization (ESI), Mass spectrometry (including tandem mass spectrometry, eg LC MS/MS), biosensor technology, evanescent fiber-optics technology or DNA chip technology, can also be employed.
  • MALDI-TOF matrix-assisted laser deso ⁇ tion/ionization time of flight
  • ESI electrospray ionization
  • Mass spectrometry including tandem mass spectrometry, eg LC MS/MS
  • biosensor technology evanescent fiber-optics technology or DNA chip technology
  • the enhanced methylation is detected by performing a process comprising:
  • probes and primers comprise about 1-5 CpG islands that are specifically methylated in colorectal cancer cell lines and/or Stage (C) colorectal cancer patient samples.
  • Shorter probes or primers, sequence variants that comprise the CpG island regions of these probes, and other functionally-equivalent probes are also contemplated for use in the inventive diagnostic assays described herein.
  • Other probe/primer combinations, and other primer combinations are also encompassed by the present invention.
  • the assay format comprises a negative read-out system in which reduced methylation of DNA from a healthy/normal control sample is detected as a positive signal and preferably, methylated DNA from a colorectal cancer sample is not detected or is only weakly detected.
  • the reduced methylation is determined using a process comprising: (i) treating the nucleic acid with an amount of a compound that selectively mutates a non-methylated cytosine residue within a CpG island under conditions sufficient to induce mutagenesis thereby producing a mutated nucleic acid; (ii) hybridizing the nucleic acid to a probe or primer comprising a nucleotide sequence that is complementary to a sequence comprising the mutated cytosine residue under conditions such that selective hybridization to the mutated nucleic acid occurs; and (iii) detecting the selective hybridization.
  • the term “selective hybridisation” means that hybridisation of a probe or primer to the mutated nucleic acid occurs at a higher frequency or rate, or has a higher maximum reaction velocity, than hybridisation of the same probe or primer to the corresponding non-mutated sequence.
  • the probe or primer does not hybridise to the methylated sequence under the reaction conditions used.
  • the hybridisation is detected using Southern, dot blot, slot blot or other nucleic acid hybridisation means (Kawai et al, Mol. Cell. Biol. 14, 7421-7427, 1994; Gonzalgo et al, Cancer Res. 57, 594-599, 1997).
  • Subject to appropriate probe selection, such assay formats are generally described herein above and apply mutatis mutandis to the presently described selective mutagenesis approach.
  • a ligase chain reaction format is employed to distinguish between a non- mutated and mutated MCC gene promoter.
  • the assay requirements and conditions are as described herein above for positive read-out assays and apply mutatis mutandis to the present format.
  • probes will differ.
  • combinations of probes are based upon the sequence set forth in SEQ ID NO: 24.
  • the ligated probes are detected by adding an "anchor" primer based on SEQ ID NO: 24 following the ligation reaction and performing PCR in accordance with standard amplification procedures as described herein.
  • the ligase chain reaction probe(s) have 3 '-terminal and/or 5 '-terminal sequences that comprise a CpG island that is not methylated in a healthy control subject, but is hyper methylated in colorectal cancer, such that the diagnostic probe and contiguous probe are capable of being ligated only when the cytosine of the CpG island is mutated to thymidine e.g., in the case of a non-methylated cytosine residue.
  • the diagnostic and contiguous probes are selected from the group consisting of (i) a probe comprising a sequence contained within the sequence set forth in SEQ ID NO: 24 and having a 5 '-terminal guanosine residue at a position within SEQ ID NO: 24 selected from the group consisting of position 293, position 298, position 303, position 339, position 342, position 348, position 360, position 362, position 365, position 367, position 373, position 395 and position 399; (ii) a probe comprising a sequence contained within the sequence set forth in SEQ ID NO: 24 and having a 3 '-terminal thymidine residue at a position within SEQ ID NO: 24 selected from the group consisting of position 292, position 297, position 302, position 338, position 341, position 347, position 359, position 361, position 364, position 366 position 372, position 394 and position 398; (iii) a probe comprising a sequence that is complementary to a portion of SEQ ID NO: 24 and having
  • exemplary combinations of diagnostic and contiguous probes that are ligatable are selected by combination of a probe set forth in sub-paragraph (i) supra with a probe set forth in sub-paragraph (ii) supra, or alternatively, by combination of a probe set forth in sub-paragraph (iii) supra with a probe set forth in sub-paragraph (iv).
  • multiple sets of diagnostic probes and multiple sets of contiguous probes can be employed to provide for the detection of mutated sequences in the normal/healthy control that would otherwise be protected from mutation by methylation in colorectal cancer. Again, the selection of these probes can be based upon the criteria for probe design described in the preceding paragraphs.
  • the hybridization is detected using an amplification system using any amplification assay format as described herein above for positive read-out assay albeit using primers (and probes where applicable) that are derived from the nucleotide sequence set forth in SEQ ID NO: 24.
  • mutation of non-methylated cytosine residues within the CpG islands of the MCC gene promoter of a healthy/normal subject is enhanced relative to the colorectal cancer sample.
  • This enables enhanced annealing of one or more primers derived from SEQ ID NO: 24 to the MCC gene promoter in the bisulphite-treated control sample thereby producing an enhanced signal relative to a colorectal cancer sample.
  • the primers are selected from the group consisting of (i) a primer comprising a sequence contained within residues from position 284 to position 304 of SEQ ID NO: 24; (ii) a primer comprising a sequence contained within residues from position 335 to position 355 of SEQ ID NO: 24; (iii) a primer comprising a sequence contained within residues from position 361 to position 382 of SEQ ID NO: 24; (v) a primer comprising a sequence contained within residues from position 383 to about position 404 of SEQ ID NO: 24; and (v) a primer comprising a sequence that is complementary to any one of (i) to (iv).
  • Amplification primers will preferably comprise sequences corresponding or complementary to any one or more of SEQ ID Nos: 25-30.
  • the nucleotide sequence set forth in SEQ ID NO: 25 corresponds to residues at positions 284 to about 304 of SEQ ID NO: 24 in the 5 -untranslated region of a MCC gene promoter from a healthy/normal control following mutagenesis using bisulphite.
  • the sequence set forth in SEQ ID NO: 26 corresponds to residues at positions 335 to 355 of SEQ ID NO: 24 in the 5 -untranslated region of a MCC gene promoter from a healthy/normal control following mutagenesis using bisulphite.
  • sequence set forth in SEQ ID NO: 27 corresponds to the complement of residues at positions 361 to 382 of SEQ ID NO: 18 in the 5 -untranslated region of a MCC gene promoter from a healthy/normal control following mutagenesis using bisulphite.
  • sequence set forth in SEQ ID NO: 28 corresponds to residues at positions 335 to 354 of SEQ ID NO: 24 in the 5 -untranslated region of a MCC gene promoter from a healthy/normal control following mutagenesis using bisulphite.
  • sequence set forth in SEQ ID NO: 29 corresponds to residues at positions 359 to 377 of SEQ ID NO: 24 in the 5 -untranslated region of a MCC gene promoter from a healthy/normal control following mutagenesis using bisulphite.
  • sequence set forth in SEQ ID NO: 30 corresponds to the complement of residues at positions 383 to 404 of SEQ ID NO: 18 in the 5 -untranslated region of a MCC gene promoter from a healthy/normal control following mutagenesis using bisulphite. Accordingly, these primers are derived from the sequence of a non-hyper methylated MCC gene promoter that has been mutated using bisulphite.
  • the primers used will be a combination selected from the group consisting of: (i) a primer comprising the sequence set forth in SEQ ID NO: 25 and a primer comprising a sequence that is the complement of SEQ ID NO: 26; (ii) a primer comprising the sequence set forth in SEQ ID NO: 25 and a primer comprising the sequence set forth in SEQ ID NO: 27;
  • the detection step of the assay formats described herein clearly encompass the use of multiple rounds of amplifications and/or combinations of amplification and classical nucleic acid hybridization steps, in any order.
  • nucleic acid of the MCC gene promoter using a combination of a primer comprising the sequence set forth in SEQ ID NO: 28 and a primer comprising the sequence of SEQ ID NO: 30, and then to hybridize the amplified nucleic acid using a labeled hybridization probe that comprises the nucleotide sequence of SEQ ID NO: 29 or a complementary sequence thereto.
  • a labeled hybridization probe that comprises the nucleotide sequence of SEQ ID NO: 29 or a complementary sequence thereto.
  • a first series of amplification reactions may employ a primer comprising the sequence set forth in SEQ ID NO: 25 and a primer comprising the sequence of SEQ ID NO: 27, and a second series of amplification reactions may employ: (i) a primer comprising the sequence set forth in SEQ ID NO: 25 and a primer comprising a sequence that is complementary to the sequence of SEQ ID NO: 26; or alternatively, (ii) a primer comprising the sequence set forth in SEQ ID NO: 26 and a primer comprising the sequence of SEQ ID NO: 27; or alternatively, (iii) a primer comprising the sequence set forth in SEQ ID NO: 26 and a primer comprising the sequence of SEQ ID NO: 28; or alternatively, (iv) a primer comprising the sequence of SEQ ID NO: 26 and a primer comprising the a sequence that is complementary to SEQ ID
  • a second series of amplification reactions may employ any one or more of the sequences set forth in SEQ ID Nos: 26-29 in an appropriate combination as described supra to amplify the intervening region there between.
  • the performance of each and every of the above-mentioned second series of amplification reactions simultaneously or contemporaneously is also encompassed by the present invention.
  • a combination of a primer comprising the sequence set forth in SEQ ID NO: 28 and a primer comprising the sequence of SEQ ID NO: 30 is used for amplification, and the amplified nucleic acid is hybridized to a labeled hybridization probe that comprises the nucleotide sequence of SEQ ID NO: 29 or a complementary sequence thereto.
  • hybridization probes capable of hybridizing to the amplified nucleic acid
  • the probe comprises a nucleotide sequence that occurs within the MCC gene promoter at a position between the two amplification primer sequences and preferably does not comprise a sequence of either amplification primer used.
  • the skilled artisan will readily be capable of determining the nucleotide sequence of a suitable hybridization probe to perform this embodiment based upon the disclosure in SEQ ID NO: 24 and, as a consequence, the present invention is not to be limited by the precise sequence of a hybridization probe used in conjunction with amplification primers as described.
  • a TaqMan probe comprising the sequences set forth in SEQ ID NOs: 26 or 29 labelled at its 5'- and 3'- ends with different fluorescent ligands, e.g., TAMRA and/or FAM and/or a BHQ.
  • the probe is labelled at the 5'-end using FAM and at the 3 '-end using TAMRA.
  • Such a probe is generally used in conjunction with amplification primers having different and non-overlapping sequences with SEQ ID NOs: 26 or 29 in the MCC gene promoter, as described herein above.
  • the amplification primers set forth in SEQ ID Nos: 25 and 27 are used in conjunction with a TaqMan probe comprising the nucleotide sequence of SEQ ID NO: 26.
  • the amplification primers set forth in SEQ ID Nos: 28 and 30 are used in conjunction with a TaqMan probe comprising the nucleotide sequence of SEQ ID NO: 29.
  • the reduced methylation in the normal/healthy control subject is detected by performing a process comprising:
  • nucleic acid treating the nucleic acid with an amount of a compound that selectively mutates non-methylated cytosine residues under conditions sufficient to induce mutagenesis thereby producing a mutated nucleic acid; (ii) hybridizing the nucleic acid to two non-overlapping and non-complementary primers each of which comprises a nucleotide sequence that is complementary to a sequence in the DNA comprising a mutated cytosine residue under conditions such that hybridization to the mutated nucleic acid occurs;
  • the present invention provides multiplexed assays comprising the detection of hypermethylation in the MCC gene promoter in combination with the detection of hypermethylation within a gene selected from the group consisting of HLTF, APC, pKf ⁇ pl ⁇ , HPP1, hMLHl, MGMT, and combinations thereof.
  • this embodiment provides for the detection of hyper methylation in the MCC gene promoter in combination with a determination of a a CpG island methylation phenotype (CIMP) of the sample.
  • CIMP CpG island methylation phenotype
  • this embodiment provides for the detection of hyper methylation in the MCC gene promoter in combination with a determination of a mutation within the DCC gene and/or APC gene.
  • Detection of methylated CpG sites within the 5' region of the pl6 m ⁇ 4a gene is preferably performed using MS PCR essentially as described by Clark et al, Nucleic Acids Res. 22, 2990-2997, 1994 and/or Herman et al, Proc. Natl. Acad. Sci. USA, 93, 9821-9826, 1996. Briefly, about 1.0 ⁇ g of purified DNA is diluted in 0.3N NaOH and DNA denatured at 37°C for about 15 min. The sample is then treated with sodium bisulfite solution (pH 5.0) and hydroquinone prepared fresh for each analysis.
  • Samples are incubated at about 55 °C for about 16 h, in the presence of an overlay of mineral oil to prevent evaporation.
  • the solution is then cooled to -80°C for about 10 min, the mineral oil removed, and bisulfite-modified DNA purified e.g., using the Wizard DNA Clean-UP System and Vacuum Manifold (Promega) according to the manufacturer's instructions.
  • Cytosine-to-uracil conversion is achieved using alkali (e.g., 0.3N NaOH) at 37°C for about 15 min.
  • a methylation-positive colorectal cancer cell line e.g., SW- 480; American Type Culture Collection
  • a methylation-negative cell line e.g., SK-N-SH; American Type Culture Collection
  • PCR reactions as with assays for MCC promoter methylation or methylation of any other gene described herein.
  • bisulfite-treated DNA is amplified using commercially available primers (Life Technologies, Inc.) specific for methylated (M primers) and nonmethylated (U primers) CpG sites within the p!6 mK4a promoter region; (M primers: 5 - TTATTAGAGGGTGGGGCGGATCGC (SEQ ID NO: 35) and 5 - GACCCCGAACCGCGACCGTAA (SEQ ID NO: 36); and U primers: 5 - TTATTAGAGGGTGGGGTGGATTGT (SEQ ID NO: 37) and 5 - CCACCTAAATCAACCTCCAACCA (SEQ ID NO: 38).
  • M primers 5 - TTATTAGAGGGTGGGGCGGATCGC (SEQ ID NO: 35) and 5 - GACCCCGAACCGCGACCGTAA (SEQ ID NO: 36)
  • U primers 5 - TTATTAGAGGGTGGGGTGGATTGT (SEQ ID NO: 37) and 5 - CCACCTAAATC
  • Standard PCR reaction conditions are used (e.g., GeneAmp PCR buffer, Perkin-Elmer Co ⁇ .; 1.5 mM MgCl 2; 200 ⁇ M each deoxynucleotide triphosphate; 0.4 ⁇ M each primer; 50ng modified DNA template; and 2.5 units of AmpliTaq, Perkin-Elmer Co ⁇ . in a total volume of 50 ⁇ l; preheated at 94°C for 1 min, then 30-35 cycles of 94°C for 30 s, 65°C for 10-30 s, 72°C for 30 s, and a final extension at 72°C for 10 min).
  • Methods used to determine methylation of pl6 ARF14 are also suitable for determining the methylation of other genes multiplexed with MCC.
  • Detection of methylated CpG sites within the 5' region of the HPP1 gene is preferably performed using MS PCR essentially as described above using the primers: 5 - GTTATCGTCGTCGTTTTTGTTGTC-3' (SEQ ID NO: 39) and 5 - GACTTCCGAAAAACACAAAATCG-3' (SEQ ID NO: 40).
  • the amplification product is preferably detected by hybridizing a probe comprising the sequence 5 - 6FAM-CCGAACAACGAACTACTAAACATCCCGCG-TAMRA -3' (SEQ ID NO: 41) to the amplification p'roduct.
  • Detection of methylated CpG sites within the 5' region of the hMLHl gene is preferably performed using MS PCR essentially as described above using the primers:
  • the amplification product is preferably detected by hybridizing a probe comprising the sequence 5 -6FAM-CGAACGCGACGTCAAACGCCACTA-TAMRA -3* (SEQ ID NO: 44) to the amplification product.
  • Detection of methylated CpG sites within the 5' region of the O ⁇ -Methylguanine-DNA methyltransferase (MGMT) gene is preferably performed using MS PCR essentially as described above using the primers: 5 -TTTCGACGTTCGTAGGTTTTCGC-3 (SEQ ID NO: 45) and 5 - GCACTCTTCCGAAAACGAAACG-3' (SEQ ID NO: 46).
  • the amplification product is preferably detected by hybridizing a probe to the amplification product.
  • MCA essentially as described by Toyota et al, Cancer Res. 59, 2307-2312, 1999. Briefly, a fragment of genomic DNA is digested with the methylation sensitive Sw ⁇ l enzyme to remove unmethylated Smal sites, and produce a blunt ended fragment. Methylated Sw ⁇ l sites are then digested with the nonmethylation-sensitive isoschizomer Xmal, which digests methylated CpG islands within Sw ⁇ l recognition sites, leaving a CCGG overhang. Adaptors are ligated to these overhangs, and PCR is performed to amplify the methylated sequences.
  • the MCA amplicons are hybridizaed to one or more MINT probes e.g., in a dot blot analysis.
  • MINT1, MINT2, MINT12 and MINT 31 probes are employed.
  • signals are generated using at least 3 or 4 MINT probes.
  • HLTF gene shall be taken to mean a nucleic acid, including any genomic gene, that is linked to or positioned at map position 3q24-q25 of the human genome, or any mRNA transcript thereof, or any genomic gene or mRNA transcript from a human or non-human animal that comprises a nucleotide sequence having at least about 80% identity to the sequence of a human HLTF gene transcript as set forth in SEQ ID NO: 4 or encoding a polypeptide having an amino acid sequence that is at least about 60% identity to a sequence as set forth in SEQ ID NO: 5 or 6 and preferably exhibits an activity characteristic of HLTF by virtue of HLTF belonging to the SWI/SNF family of transcriptional activators.
  • SEQ ID NO: 4 the nucleotide sequence of the open reading frame, 5 -untranslated region and 3 -untranslated region of the HLTF gene (Ding et al, DNA Cell Biol 15, 429-442, 1996, inco ⁇ orated herein by reference) is set forth herein as SEQ ID NO: 4. It is known that there are two HLTF proteins encoded by SEQ ID NO: 4 which are the products of alternate translation initiation.
  • DCC gene or “CRC18 gene” or “CRCR1 gene” shall be taken to mean a nucleic acid, including any genomic gene, that is linked to or positioned at map position 18q21.3 of the human genome, or any mRNA transcript thereof, or any genomic gene or mRNA transcript from a human or non-human animal that comprises a nucleotide sequence having at least about 80% identity to the sequence of a human DCC gene transcript as set forth in SEQ ID NO: 7 or encoding a polypeptide having an amino acid sequence that is at least about 60% identity to a sequence as set forth in SEQ ID NO: 8.
  • nucleotide sequence of the open reading frame and 3 -untranslated region of the human DCC gene (O'Boyle et al, Cancer Invest. 21, 484-485, 2003; Bamias et al, Cancer Invest. 21, 333-340, 2003; Hedrick et al, Genes Devel 8, 1174-1183, 1994; Cho et al, Genomics 19, 525-531, 1994; and Fearon et al, Science 247, 49-56, 1990 each of which is inco ⁇ orated herein by reference) is set forth herein as SEQ ID NO: 7.
  • amino acid sequence of the corresponding encoded DCC polypeptide is set forth as SEQ ID NO: 8.
  • the term "APC gene” shall be taken to mean a nucleic acid, including any genomic gene, that is linked to or positioned at map position 5q21 of the human genome, or any mRNA transcript thereof, or any genomic gene or mRNA transcript from a human or non-human animal that comprises a nucleotide sequence having at least about 80% identity to the sequence of a human APC gene transcript as set forth in SEQ ID NO: 9 or encoding a polypeptide having an amino acid sequence that is at least about 60% identity to a sequence as set forth in SEQ ID NO: 10.
  • nucleotide sequence of the 5 -untranslated region, open reading frame, and 3 -untranslated region of the human APC gene is set forth herein as SEQ ID NO: 9.
  • amino acid sequence of the corresponding encoded APC polypeptide is set forth as SEQ ID NO: 10.
  • Any multiplex/multianalyte assay format described herein that involve detection of MCC hypermethylation will enhance the rate of positive detection of early stage colorectal cancer to about 80% or higher, and strengthen the predictive value of tests for determining a malignant potential of a polyp, especially HP.
  • probes and primers specific for genes other than MCC are combined with primers and probes for MCC in single reactions to identify diagnostic methylation patterns in both gene promoters simultaneously.
  • the only requirement for such approaches is that the probes/primers are separately labelled or otherwise separately detectable.
  • reactions are performed separately using the same samples as starting material.
  • the detection of hyper methylation within the MCC gene promoter is combined with detection of mutations within the APC gene essentially as described in U.S. Pat. No. 5,648,212 and /or by Traverso et al, New England J. Med 346, 3 -320, 2002.
  • the detection of hyper methylation within the MCC gene promoter is combined with detection of mutations within the DCC gene.
  • the present invention clearly encompasses any and all combinations of such multiplex assay formats.
  • suitable samples include bodily fluids and stool samples.
  • DNA can be prepared readily from all such samples using standard procedures.
  • stool samples are known to contain various inhibitors of DNA polymerase and, as a consequence, it is preferred to capture the DNA from such samples onto magnetic beads coated with suitable probes to permit amplification of a hyper methylated region of the MCC gene promoter e.g., between nucleotide positions 284 and 404 of SEQ ID Nos: 3, 17 or 24.
  • the isolation of DNA from stool samples is described by Traverso et al., for amplification of the APC gene between codons 1210 and 1581.
  • Bodily fluids shall be taken to include whole blood, serum, peripheral blood mononuclear cells (PBMC), buffy coat fraction, urine, semen, or abdominal fluid.
  • PBMC peripheral blood mononuclear cells
  • the biological sample will comprise colorectum tissue derived preferably by colonoscopy, or cells, and those tissues known to comprise cancer cells arising from a micrometastasis of a colorectal cancer.
  • cancer cell includes any biological specimen or sample comprising a cancer cell irrespective of its degree of isolation or purity, such as, for example, tissues, organs, cell lines, bodily fluids, or histology specimens that comprise a cell in the early stages of transformation or having been transformed.
  • cancer cell is not to be limited by the stage of a cancer in the subject from which said cancer cell is derived (ie. whether or not the patient is in remission or undergoing disease recunence or whether or not the cancer is a primary tumor or the consequence of metastases).
  • cancer cell is not to be limited by the stage of the cell cycle of said cancer cell.
  • the biological sample has been isolated previously from the subject. In such cases, the sample may be processed or partially processed into a nucleic acid sample that is substantially free of contaminating protein. All such embodiments are encompassed by the present invention.
  • the diagnostic method provided by the present invention involves a degree of quantification to determine elevated or enhanced methylation of nucleic acid in tissue that is suspected of comprising a colorectal tumour cell or metastases thereof. Such quantification is readily provided by the inclusion of appropriate control samples in the assays as described below.
  • control when internal controls are not included in each assay conducted, the control may be derived from an established data set.
  • Data pertaining to the control subjects are selected from the group consisting of: 1. a data set comprising measurements of the degree of methylation for a typical population of subjects known to have colorectal cancer; 2. a data set comprising measurements of the degree of methylation for the subject being tested wherein said measurements have been made previously, such as, for example, when the subject was known to healthy or, in the case of a subject having colorectal cancer, when the subject was diagnosed or at an earlier stage in disease progression; 3. a data set comprising measurements of the degree of methylation for a healthy individual or a population of healthy individuals;
  • a data set comprising measurements of the degree of methylation for a normal individual or a population of normal individuals
  • a data set comprising measurements of the degree of methylation from the subject being tested wherein the measurements are determined in a matched sample.
  • the term "typical population” with respect to subjects known to have colorectal cancer shall be taken to refer to a population or sample of subjects diagnosed with colorectal cancer that is representative of the spectrum of colorectal cancer patients. This is not to be taken as requiring a strict normal distribution of mo ⁇ hological or clinicopathopathological parameters in the population, since some variation in such a distribution is permissible.
  • a "typical population” will exhibit a spectrum of colorectal cancers at different stages of disease progression and with tumours at different stages and having different mo ⁇ hologies or degrees of differentiation. It is particularly preferred that a "typical population” exhibits the expression characteristics of a cohort of subjects or non-cancerous cell lines as described herein.
  • the term "healthy individual” shall be taken to mean an individual who is known not to suffer from colorectal cancer, such knowledge being derived from clinical data on the individual, including, but not limited to, a different colorectal cancer diagnostic assay to that described herein.
  • the present invention is particularly useful for the early detection of colorectal cancer or survival, it is preferred that the healthy individual is asymptomatic with respect to the any symptoms associated with colorectal cancer.
  • normal individual shall be taken to mean an individual having a normal level of methylation or expression of a colorectal cancer-associated gene or a normal level of expression of a colorectal cancer-associated protein in a particular sample derived from said individual.
  • data obtained from a sufficiently large sample of the population will normalize, allowing the generation of a data set for determining the average level of a particular parameter.
  • the level of methylation or expression of a colorectal cancer-associated gene or the level of expression of a colorectal cancer-associated protein can be determined for any population of individuals, and for any sample derived from said individual, for subsequent comparison to levels determined for a sample being assayed.
  • internal controls are preferably included in each assay conducted to control for variation.
  • matched sample shall be taken to mean that a control sample is derived from the same subject as the test sample is derived, at approximately the same point in time.
  • the control sample shows little or no mo ⁇ hological and/or pathological indications of colorectal cancer. Matched samples are not applicable to blood-based or serum-based assays.
  • the matched sample is a region of a colon, colon wall or lining, abdominal cavity, derived from a subject suffering from colorectal cancer, wherein that sample does not include malignant cells or exhibit any symptom of the disease (e.g., no detectable polyposis, carcinoma in situ, tumor or metastases as determined by art-recognized methods such as, for example, biopsy of resected colon).
  • the sample comprises less than about 20% malignant colorectal cells, more preferably less than about 10% malignant colorectal cells, even more preferably less than about 5% malignant colorectal cells and most preferably less than about 1% malignant colorectal cells.
  • Mo ⁇ hological and pathological indications of malignant colorectal cells are known in the art and/or described herein.
  • a further aspect of the present invention provides an isolated nucleic acid probe or primer that is capable of selectively hybridising to a region of the MCC gene promoter that is hyper methylated in a colorectal cancer, wherein said region comprises or is contained within nucleotide residues from about position 284 to about position 404 of SEQ ID NO: 3 or SEQ ID NO: 17 or SEQ ID NO: 24.
  • the diagnostic probe and the contiguous probe are not already available, they must be synthesized. Apparatus for such synthesis is presently available commercially, such as the Applied Biosystems 380A DNA synthesizer and techniques for synthesis of various nucleic acids are available in the literature.
  • the probes are prepared for ligation, e.g., if ligase is to be used, the probe which will have its 5' end adjacent the 3' end of the other probe when hybridized to the sample nucleic acid is phosphorylated in order to later be able to form a phosphodiester bond between the two probes.
  • One of the probes is then labeled. This labeling can be done as part of the phosphorylation process above using radioactive phosphorus, or can be accomplished as a separate operation by covalently attaching chromophores, fluorescent moieties, enzymes, antigens, chemiluminescent moieties, groups with specific binding activity, or electrochemically detectable moieties, etc. such as, for example, using T4 polynucleotide kinase.
  • preferred hybridisation probes are selected from the group consisting of
  • (x) a probe of at least about 18 nucleotides in length and comprising a sequence that is complementary to a portion of SEQ ID NO: 24 and having a 5 '-terminal guanosine residue complementary to a cytosine residue within SEQ ID NO: 24 at a position selected from the group consisting of position 292, position 297, position 302, position 338, position 341, position 347, position 359, position 361, position 364, position 366 position 372, position 394 and position 398;
  • xi a probe of at least about 18 nucleotides in length and comprising a sequence that is complementary to a portion of SEQ ID NO: 24 and having a 3 '-terminal cytosine residue complementary to a guanosine residue within SEQ ID NO: 24 at a position selected from the group consisting of position 293, position 298, position 303, position 339, position 342, position 348, position 360, position 362, position 365, position 367, position 373, position 395 and position 399; (xii) a probe comprising a sequence that is complementary to any one of (i) to (xi).
  • Particularly preferred amplification primers comprise a nucleotide sequence selected from the group consisting of SEQ ID Nos: 11, 12, 13, 14, 15, 16, 18, 19, 20, 21, 22, 23, 25, 26, 27, 28, 29 and 30, and sequences complementary thereto.
  • Example 1 Loss of expression of the MCC gene in colon cancer correlates with hypermethylation of the MCC gene promoter
  • the MCC promoter region containing a CpG island was amplified using primers MCC-promF 5'- GGTTAGTAGTTAGATAGTTGT-3' (SEQ ID NO: 31), and MCC-promR 5'- TACTTAATCCCTTCTACCAC-3' (SEQ ID NO: 32).
  • PCR conditions were 95°C (30s), 53°C (45s), 72°C (90s) for 40 cycles with an initial denaturation at 95°C for 12 min and final elongation at 72°C for 7min. All PCR reactions in this study were carried out using AmpliTaq Gold (Applied Biosystems) in a DNA engine DYADTM (MJ Research).
  • a 514-bp fragment was detected in all cell lines and sequenced using BigDye on an ABI PRISM 3700 DNA Analyzer (Applied Biosystems).
  • a densely methylated section of the MCC promoter was amplified from bisulphite- treated patient specimens and cell line DNA using methylation specific primers MCC- metFl 5'-TATTGTTTCGGAACGGGGCGT-3' (SEQ ID NO: 18) and MCC-metRl 5'-CAAAAAACTCGATAACGCGACG-3' (SEQ ID NO: 20).
  • PCR conditions were 95°C (30s), 61°C (45s), 72°C (30s) for 35 cycles.
  • the cycling conditions included an initial denaturation step at 95°C for 12min and final elongation at 72°C for 7min.
  • the predicted fragment size of the amplification product was about 99bp.
  • MYOD1 gene primers were used to amplify a MYOD1 gene fragment (Usadel et al, Cancer Res. 62(2), 371-375, 2002) in a region of the gene that does not contain any CpG nucleotide sequences. Accordingly, this control reaction ensured integrity of each DNA specimen.
  • PCR conditions were 95°C (45s), 57°C (45s), 72°C (60s) for 40 cycles.
  • the pi 6 gene promoter was amplified using primers for the methylated sequences as previously described (Herman et al, Pre. Natl Acad. Sci. USA 93, 9821-9826, 1996).
  • PCR conditions included 40 cycles of amplification wherein each cycle consisted of 95°C for 30 s, 65°C for 30 s, and 72°C for 30 s. These 40 cycles were flanked by an initial denaturation step at 95°C for 12 min, and a final elongation step at 72°C for 7 min.
  • PCR reactions were carried out using AmpliTaq Gold (Applied Biosystems) in a DNA engine DYAD (MJ Reasearch).
  • the primers MCC-metFl and MCC-metRl were also used in a Real-time PCR together with the methylated amplicon-specific fluoro genie hybridisation probe FAM5'- TTTCGTCGTTGTCGTAGTTGC-3 'TAMRA (SEQ ID NO: 20).
  • the PCR was carried out using the TaqMan PCR buffer A pack (Applied Biosystems) in a Rotorgene 3000 (Corbett Research).
  • TaqMan assays are performed using the amplification primers set forth in SEQ ID Nos: 21 and 23, with a TaqMan probe based upon the nucleotide sequence set forth in SEQ ID NO: 22, appropriately labeled at the 5 -end using FAM and at the 3 -end using BHQ-1.
  • the PCR was carried out using the REalMasterMix Probe ROX (Eppendorf) with 4mM MgCl 2 in a Rotorgene 3000 (Corbett Research). PCR conditions included an initial denaturation at 95°C for 2 min, followed by 50 cycles wherein each cycle consisted of 95°C for 15 s and 60°C for 60 s.
  • a housekeeping gene ALAS-1 was used for an internal reference PCR (422bp fragment) to test for the integrity of the cDNA specimens in the same conditions.
  • a MCC cDNA sequence obtained from GenBank (BC009279) was amended and extended 5' using further sequence data from the Ensembl Genome Browser located at the URL ensemble.org. The sequence discrepancy involved nucleotides 1-7 of the GenBank entry.
  • a 403 -bp section of the MCC promoter (-542 to -139) was found to contain 48 CpG sites (Fig. 1).
  • Bisulphite sequencing of the promoter revealed extensive methylation in six colon cancer cell lines, Lisp 1, Co-115, LS513, KM12SM, LS411N, HCT15.
  • the majority of the CpG sites in the other cell lines studied were bisulphite-converted, except for six sites in the middle of the CpG island. These six sites showed a partial or a complete lack of bisulphite conversion, which also affected the other five C nucleotides in this region (-354 to -320; Fig 2).
  • One further CpG site (-269) outside this region showed partial methylation in six of the eight "unmefhylated" cell lines.
  • cD ⁇ A was prepared from three cell lines HCTl 5, LS41 IN and TOV21G.
  • RT-PCR was carried out using a primer set in exons 1 and 2.
  • the predicted 265-bp fragment was consistently amplified in the cell line TOV21 G that showed no methylation of the MCC promoter (Fig 3).
  • No fragment was detectable in the cell line HCTl 5, whereas the control housekeeping gene ALAS-1 amplified normally.
  • a MCC cDNA fragment was detectable in the cell line LS411N but with much lower intensity compared with the control ALAS-1 PCR and it was not always detectable in every RT-PCR.
  • the 26 CpG sites in region -304 to -139 were the shown to be an important indicator of MCC gene silencing or reduced expression. Methylation-specific PCR.
  • Example 2 Methylation of MCC promoter sequences in hype ⁇ lastic polyps Tissue samples A total of 43 polyp samples were obtained. At the filing date, 19 samples had been classified as hype ⁇ lastic polyp or adenoma. Seven hype ⁇ lastic polyps were analyzed, at least two of which were classified as variant HPs. Twelve adenomas were also analyzed. Methylation specific real-time PCR.
  • the primers MCC-metF2 (SEQ ID NO: 19) and MCC-metR2 (SEQ ID NO: 23) were used in a real-time PCR together with the methylated amplicon-specific fluorogenic hybridisation probe 5'-6FAM-CGCGTCGCGTTATCGAGTT-BHQ-l -3' (SEQ ID NO: 22).
  • the PCR was carried out using the RealMasterMix Probe ROX (Eppendorf) with 4mM Mg 2+ in a Rotorgene 3000 (Corbett Research). PCR conditions were 95°C (15s), 60°C (60s) for 50 cycles, with an initial denaturation at 95°C for 2min.
  • APC gene promoter methylation and pi 6 gene promoter methylation were also analysed in these specimens.
  • the pi 6 gene promoter was amplified using primers for the methylated sequences and PCR conditions as described herein above (e.g., Example 1 and Herman et al, Proc. Natl acad. Sci. USA 93, 9821-9826, 1996).
  • Methylated APC gene promoter sequences were amplified using the PCR primers and fluorogenic hybridization TaqMan probe as described by Usadel et al, Cancer Res. 62, 371-175, 2002).
  • Real-time PCRs were carried out using the real MasterMix Probe ROX (Eppendorf) in a Rotorgene 3000 (Corbett Research). PCR conditions were 95°C (15s), 60°C (60s) for 50 cycles, with an initial denaturation at 95°C for 2min.
  • MCC, APC and pi 6 methylation data are summarized in Figure 9. Those data show that methylation of MCC was found with either pi 6 methylation and/or APC methylation in all HPs examined, and that methylation of MCC or pi 6 or APC occurred in 11 of the 12 adenomas tested. TABLE 2 MCC methylation in early stage of colon carcinogenesis

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Abstract

La présente invention a trait à des dosages pour le diagnostic de cancer colorectal ou d'une prédisposition à cette maladie ou du potentiel néoplasique d'un polype colorectal dans lequel on détermine l'hyperméthylation d'un promoteur de gène MCC. L'invention a trait à divers tests diagnostiques d'analytes multiples et de réactifs pour la réalisation de ces dosages de diagnostic/prévision.
PCT/AU2005/000077 2004-01-23 2005-01-24 Procede de diagnostic de cancer colorectal et reactifs de mise en oeuvre WO2005071404A1 (fr)

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US10/586,978 US20070243531A1 (en) 2004-01-23 2005-01-24 Methods of Diagnosing Colorectal Cancer and Reagents Therefor
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EP2011068A4 (fr) * 2006-03-30 2010-06-23 Univ Maryland Méthylation de gènes utilisés comme prédicteur de la formation et de la réapparition de polypes
DE102017125013A1 (de) * 2017-10-25 2019-04-25 Epiontis Gmbh MCC als epigenetischer Marker zur Identifizierung von Immunzellen, insbesondere basophiler Granulozyten

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US5330892A (en) * 1991-03-13 1994-07-19 The Johns Hopkins University MCC gene (mutated in colorectal cancer) used for diagnosis of cancer in humans
AU2002951346A0 (en) * 2002-09-05 2002-09-26 Garvan Institute Of Medical Research Diagnosis of ovarian cancer

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WILSON R.H. ET AL: "Molecular Genetics of Colorectal Cancer (part 1)", CLINICAL ONCOLOGY, vol. 9, 1997, pages 14 - 19, XP008074248 *

Cited By (5)

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Publication number Priority date Publication date Assignee Title
EP2011068A4 (fr) * 2006-03-30 2010-06-23 Univ Maryland Méthylation de gènes utilisés comme prédicteur de la formation et de la réapparition de polypes
DE102017125013A1 (de) * 2017-10-25 2019-04-25 Epiontis Gmbh MCC als epigenetischer Marker zur Identifizierung von Immunzellen, insbesondere basophiler Granulozyten
WO2019081584A1 (fr) 2017-10-25 2019-05-02 Epiontis Gmbh Utilisation du mcc comme marqueur épigénétique pour l'identification de cellules immunitaires, en particulier de granulocytes basophiles
DE102017125013B4 (de) * 2017-10-25 2019-10-17 Epiontis Gmbh MCC als epigenetischer Marker zur Identifizierung von Immunzellen, insbesondere basophiler Granulozyten
US11753683B2 (en) 2017-10-25 2023-09-12 Precision For Medicine Gmbh MCC as epigenetic marker for the identification of immune cells, in particular basophil granulocytes

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